Trigger Factor and its involvement ... - University of Otago

Trigger Factor and its involvement

in the repair of Photosystem II

Simon Tuohy

a thesis submitted for the degree of

Master of Science

at the University of Otago, Dunedin,

New Zealand.

May 2009

Abstract

Replacement of the photosynthetic D1 protein is essential for continuation

of photosynthesis and the life of a plant or cyanobacteria. Trigger Factor

(TF) is an approximately 52 kDa molecular chaperone which may bind the

ribosome-D1 complex prior to the association with the thylakoid targeted

Signal Recognition Particle (cpSRP54). Trigger Factor binds to nearly all

ribosomes, hence it is feasible that TF binds D1 protein and stalls until

cpSRP54 can target it to SecY. The aim of this research was to create a

TFSpec mutant of Synechocystis sp. PCC 6803 which would allow the char-

acterisation of the TFSpec phenotype and to characterise the Synechocystis

sp. PCC 6803 TF protein. The TFSpec strain showed several physiological

differences from Wild Type (WT). The rate of O2 evolution by the TFSpec

strain was slower than for the WT strain over the same period, and the

growth rates of the TFSpec strain was lower than the WT strain. Trigger

Factor was also differentially detected in various cellular fractions when

pelleted with ribosomes purified from cells grown under different light con-

ditions. Biophysical analysis of TF was done by Circular Dichroism and

Dynamic Light Scattering to determine the structural similarity with TF

from other species. Crystallisation of TF was also attempted. Implications

of this research are discussed.

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Acknowledgements

I would like to thank the wonderful people who have helped and encour-

aged me with this thesis. In particular my family, especially my parents,

my friends, Jessie in particular, and the awesome people of Lab 118 in the

Department of Biochemistry, Sigurd Wilbanks, Robert Fagerlund and Es-

ther Pearl. Thanks all for your support and advice on the science, writing

and life.

Simon

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Contents

1 Introduction 11.1 Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.1.1 The photosynthetic reaction . . . . . . . . . . . . . . . . . . . . 21.1.2 Photoinhibition: The Damage and Repair cycle . . . . . . . . . 4

1.2 Molecular Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.1 Trigger Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.2 The Signal Recognition Particle . . . . . . . . . . . . . . . . . . 6

1.3 E. coli experiments: What has been seen previously . . . . . . . . . . . 71.4 Membrane Protein Integration . . . . . . . . . . . . . . . . . . . . . . . 71.5 Integration of D1 into the membrane . . . . . . . . . . . . . . . . . . . 81.6 Synechocystis sp. PCC 6803 as a model . . . . . . . . . . . . . . . . . . 91.7 Research Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.7.1 Research Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Materials and Methods 132.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Chemicals and Molecular Biology Reagents . . . . . . . . . . . . . . . . 132.3 Cellular Manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.3.1 Growth and storage of E. coli and Synechocystis sp. PCC 6803strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.3.2 Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.3.3 Cyanobacterial Media . . . . . . . . . . . . . . . . . . . . . . . 172.3.4 LB media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.3.5 Media supplements and their concentrations . . . . . . . . . . . 202.3.6 Transformation of competent E. coli cells . . . . . . . . . . . . . 202.3.7 Cyanobacterial transformation . . . . . . . . . . . . . . . . . . . 21

2.4 Cellular Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.4.1 Photoautotrophic growth curves . . . . . . . . . . . . . . . . . . 222.4.2 Photoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.4.3 Chlorophyll estimation . . . . . . . . . . . . . . . . . . . . . . . 232.4.4 Oxygen electrode . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.5 DNA Isolation and Purification . . . . . . . . . . . . . . . . . . . . . . 242.5.1 Isolation of plasmid DNA . . . . . . . . . . . . . . . . . . . . . 242.5.2 Gel extraction of digested DNA . . . . . . . . . . . . . . . . . . 242.5.3 Chloroform/phenol extraction . . . . . . . . . . . . . . . . . . . 242.5.4 Ethanol precipitation . . . . . . . . . . . . . . . . . . . . . . . . 26

vii

2.5.5 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . 262.5.6 Purification of PCR products . . . . . . . . . . . . . . . . . . . 262.5.7 Primers for Polymerase Chain Reaction . . . . . . . . . . . . . . 272.5.8 Restriction endonuclease digestion . . . . . . . . . . . . . . . . . 292.5.9 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2.6 DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.6.1 Agarose gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.6.2 Quantification of DNA and proteins . . . . . . . . . . . . . . . . 312.6.3 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . 312.6.4 GateWay recombination . . . . . . . . . . . . . . . . . . . . . . 31

2.7 Southern Blot Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 312.7.1 Southern transfer blot . . . . . . . . . . . . . . . . . . . . . . . 312.7.2 Probe creation . . . . . . . . . . . . . . . . . . . . . . . . . . . 322.7.3 Pre-hybridisation and probing . . . . . . . . . . . . . . . . . . . 332.7.4 Film development . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2.8 Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.8.1 Induction of lac promoter constructs . . . . . . . . . . . . . . . 352.8.2 Lysis of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.8.3 GST resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.8.4 Talon resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.8.5 Size-exclusion chromatography . . . . . . . . . . . . . . . . . . . 372.8.6 Protein concentration . . . . . . . . . . . . . . . . . . . . . . . . 372.8.7 Ribosome and associated proteins preparation and analysis . . . 372.8.8 Ribosome Preparation . . . . . . . . . . . . . . . . . . . . . . . 38

2.9 Protein Analysis Techniques . . . . . . . . . . . . . . . . . . . . . . . . 392.9.1 Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis . 392.9.2 Electro-transfers . . . . . . . . . . . . . . . . . . . . . . . . . . 422.9.3 Western blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.9.4 Western Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . 432.9.5 ECL reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442.9.6 Antibodies used in Western Experiments . . . . . . . . . . . . . 44

2.10 Antibody Creation and Testing . . . . . . . . . . . . . . . . . . . . . . 452.10.1 Ethics approval . . . . . . . . . . . . . . . . . . . . . . . . . . . 452.10.2 Immunisation Protocol . . . . . . . . . . . . . . . . . . . . . . . 462.10.3 Dot-blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

2.11 Biophysical Characterisation . . . . . . . . . . . . . . . . . . . . . . . . 462.11.1 Dynamic light scattering . . . . . . . . . . . . . . . . . . . . . . 462.11.2 Circular dichroism . . . . . . . . . . . . . . . . . . . . . . . . . 472.11.3 Crystallisation trials . . . . . . . . . . . . . . . . . . . . . . . . 48

2.12 Computer Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

3 Creation and physiological analysis of Synechocystis sp. PCC 6803TFSpec 513.1 Creation of Synechocystis sp. PCC 6803 TFSpec . . . . . . . . . . . . . 51

3.1.1 Sequence acquisition and primer design . . . . . . . . . . . . . . 513.2 Insertion of Spectinomycin resistance into TF . . . . . . . . . . . . . . 53

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3.2.1 Transformation of cyanobacteria . . . . . . . . . . . . . . . . . . 543.3 Physiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3.3.1 Growth rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603.3.2 O2 evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.3.3 Photoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3.4 Truncations of TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643.4.1 PCR of TF domains . . . . . . . . . . . . . . . . . . . . . . . . 653.4.2 Mega-primer PCR . . . . . . . . . . . . . . . . . . . . . . . . . 673.4.3 Ligation and analysis . . . . . . . . . . . . . . . . . . . . . . . . 69

4 Antibody creation and testing 714.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714.2 Cross reactivity of TF antibodies . . . . . . . . . . . . . . . . . . . . . 72

4.2.1 Analysis of cross reactivity of the Trigger Factor antibodies . . . 724.2.2 Phylogenetic analysis of Trigger Factor proteins . . . . . . . . . 724.2.3 Analysis of TF antibodies from A. aeolicus, M. tuberculosis, E.

coli and H. influenzae . . . . . . . . . . . . . . . . . . . . . . . . 734.3 Protein expression and purification using the Gateway system . . . . . 75

4.3.1 Amplification of TF and FFH for pENTR 11 . . . . . . . . . . . 754.3.2 Recombination into pDEST vectors . . . . . . . . . . . . . . . . 76

4.4 Trial expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774.5 Large scale purification of TF with GST tag (pDEST-15) . . . . . . . . 834.6 Expression of FFH with Trx tag . . . . . . . . . . . . . . . . . . . . . . 864.7 N-terminal sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 894.8 Analysis of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

4.8.1 Analysis of TF and FFH antibodies . . . . . . . . . . . . . . . . 904.9 TF interaction with D1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

4.9.1 Ribosomal preparation . . . . . . . . . . . . . . . . . . . . . . . 924.9.2 Detection of D1 using FFH and TF . . . . . . . . . . . . . . . . 92

5 Biophysical Characterisation of Synechocystis sp. PCC 6803 TriggerFactor 955.1 Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . 955.2 Circular Dichroism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

5.2.1 Secondary Structure proportions . . . . . . . . . . . . . . . . . . 965.2.2 Dichroweb analysis . . . . . . . . . . . . . . . . . . . . . . . . . 985.2.3 TF thermal denaturation . . . . . . . . . . . . . . . . . . . . . . 985.2.4 Melting point of TF . . . . . . . . . . . . . . . . . . . . . . . . 101

5.3 Crystal Trays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

6 Discussion 1056.1 Creating the Synechocystis sp. PCC 6803 TFSpec mutant . . . . . . . . 1056.2 Phenotype of the Synechocystis sp. PCC 6803 TFSpec knockout . . . . . 106

6.2.1 Photoautotrophic growth curves . . . . . . . . . . . . . . . . . . 1066.2.2 O2 evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076.2.3 Photoinhibition studies . . . . . . . . . . . . . . . . . . . . . . . 108

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6.2.4 E. coli phenotype comparison . . . . . . . . . . . . . . . . . . . 1086.3 Truncations of TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1096.4 Analysis of non-Synechocystis sp. PCC 6803 anti-TF antibodies . . . . 111

6.4.1 Cross reactivity of antibodies . . . . . . . . . . . . . . . . . . . 1116.5 Expression of TF and FFH . . . . . . . . . . . . . . . . . . . . . . . . . 112

6.5.1 Gateway construction . . . . . . . . . . . . . . . . . . . . . . . . 1126.5.2 Trial Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 1126.5.3 Full scale expression of TF and FFH . . . . . . . . . . . . . . . 1126.5.4 N-terminal sequencing . . . . . . . . . . . . . . . . . . . . . . . 114

6.6 Analysis of anti-TF and anti-FFH antibodies . . . . . . . . . . . . . . . 1146.7 Ribosome associated TF and FFH . . . . . . . . . . . . . . . . . . . . . 115

6.7.1 Ribosomal preparation . . . . . . . . . . . . . . . . . . . . . . . 1156.7.2 Detection of FFH and TF bound to membrane associated ribosomes115

6.8 Biophysical Characterisation . . . . . . . . . . . . . . . . . . . . . . . . 1176.8.1 Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . 1176.8.2 Circular Dichroism . . . . . . . . . . . . . . . . . . . . . . . . . 1186.8.3 Crystallography setup and trials . . . . . . . . . . . . . . . . . . 119

6.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

References 121

A Creation and physiological analysis of Synechocystis sp. PCC 6803TFSpec 127A.1 TF in pGEM-t amplified with T7 sequencing primers . . . . . . . . . . 127A.2 TF in pGEM-t amplified with SP6 sequencing primers . . . . . . . . . 135A.3 TF in pENTR11 amplified with SP6 sequencing primers . . . . . . . . 144A.4 TF in pENTR11 amplified with T7 sequencing primers . . . . . . . . . 152A.5 FFH in pENTR11 amplified with SP6 sequencing primers . . . . . . . . 161A.6 FFH in pENTR11 amplified with T7 sequencing primers . . . . . . . . 169A.7 TF deletion in pGEM-5 amplified with SP6 primers . . . . . . . . . . . 176A.8 TF deletion in pGEM-5 amplified with T7 primers . . . . . . . . . . . . 185

B Antibody creation and testing 195B.1 Example trace of SEC run . . . . . . . . . . . . . . . . . . . . . . . . . 195

C Biophysical characterisation of Synechocystis sp. PCC 6803 TriggerFactor 199C.1 Tables of Crystallisation trial conditions . . . . . . . . . . . . . . . . . 199

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List of Tables

2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Molecular Biology Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.4 Enzymes used in this study . . . . . . . . . . . . . . . . . . . . . . . . 152.5 Specialty equiptment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.6 Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.7 Media Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.8 PCR template protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.9 PCR Mix 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.10 PCR Mix 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.11 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.12 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452.13 Computer Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

4.1 TF N-terminal sequencing . . . . . . . . . . . . . . . . . . . . . . . . . 894.2 FFH N-terminal sequencing . . . . . . . . . . . . . . . . . . . . . . . . 89

5.1 Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . 965.2 Dichroweb analysis: Comparisons . . . . . . . . . . . . . . . . . . . . . 985.3 Dichroweb analysis: Secondary structure proportions . . . . . . . . . . 98

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List of Figures

1.1 Photosystem II schematic . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Two main pathways for the insertion of proteins into membranes . . . . 81.3 The current picture for the integration of D1 into the thylakoid . . . . 10

3.1 Open reading Frame map of Synechocystis sll0533 . . . . . . . . . . . . 523.2 TF PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.3 Plasmid map of pGEM-T TFSpec . . . . . . . . . . . . . . . . . . . . . 553.4 TF knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.5 Colony PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583.6 Southern blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.7 Growth curves of TFSpec and WT at 30℃ and 18℃ . . . . . . . . . . . 603.8 Oxygen evolution of WT and TFSpec . . . . . . . . . . . . . . . . . . . 613.9 Photoinhibition of TFSpec vs WT . . . . . . . . . . . . . . . . . . . . . 633.10 TF truncation primer positions . . . . . . . . . . . . . . . . . . . . . . 653.11 TF domain boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 663.12 PCR of Ac-g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.1 Cross reactivity prediction of other TF antibodies . . . . . . . . . . . . 734.2 Cross reactivity of other TF antibodies . . . . . . . . . . . . . . . . . . 744.3 PCR of TF and FFH . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.4 PstI digest of pENTR-11 vector with TF and FFH insert . . . . . . . . 764.5 GateWay vector recombination . . . . . . . . . . . . . . . . . . . . . . 774.6 Trial Expression of Trx-His-TF . . . . . . . . . . . . . . . . . . . . . . 784.7 Trial Expression of GST-TF . . . . . . . . . . . . . . . . . . . . . . . . 794.8 Trial Expression of His-TF . . . . . . . . . . . . . . . . . . . . . . . . . 804.9 Trial Expression of Trx-His-FFH from the pET(gwA)-32 vector . . . . 814.10 Trial Expression of GST-FFH . . . . . . . . . . . . . . . . . . . . . . . 824.11 TF purification and column fractions . . . . . . . . . . . . . . . . . . . 844.12 TF purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854.13 Analysis of the expression and purification of FFH . . . . . . . . . . . . 874.14 Analysis of the stages of protein purification of FFH . . . . . . . . . . . 884.15 Analysis of TF antibody . . . . . . . . . . . . . . . . . . . . . . . . . . 914.16 Analysis of FFH antibody . . . . . . . . . . . . . . . . . . . . . . . . . 914.17 Analysis of ribosome preparation . . . . . . . . . . . . . . . . . . . . . 93

5.1 CD spectra of TF at 20℃ . . . . . . . . . . . . . . . . . . . . . . . . . 975.2 Thermal denaturation of TF from 5℃ to 70℃ and recovery . . . . . . . 99

xiii

5.3 Key temperatures of the thermal denaturation of TF . . . . . . . . . . 1005.4 Cross-section of the CD spectra . . . . . . . . . . . . . . . . . . . . . . 1015.5 Microcrystals in hanging drops . . . . . . . . . . . . . . . . . . . . . . . 1025.6 Precipitate in hanging drops . . . . . . . . . . . . . . . . . . . . . . . . 103

6.1 TF domain boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

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Chapter 1

Introduction

The process of photosynthesis has been analysed for decades and the finer details of the

protein components are becoming clearer. The centre of the photosynthetic complex is

assembled from an array of proteins collectively called Photosystem II (PSII), the core

of light absorption and utilisation. In the middle of this complex there is a protein

known as D1. Most of the light captured by a photosynthetic organism is funnelled

through PSII and is utilised in the process of photosynthesis. This large amount

of energy coursing through the PSII being converted from light to electrical energy

and subsequently to chemical energy, eventually causes damage to the D1 protein.

Thus D1 needs to be replaced for the continuation of the photosynthetic process. The

mechanism of integration of the D1 protein in to the thylakoid membrane of Spinacea

oleracea, examined by Zhang et al. (2001), has been proposed to occur using the Signal

Recognition Particle (SRP) pathway). Trigger Factor, a molecular chaperone, may

interact initially with the nascent D1 protein and then transfer the responsibility of

the targeting to cpSRP54, a chaperone-like moiety. Recently, it has been shown that

Escherichia coli TF does interact with the SRP when both are associated with the

ribosome (Buskiewicz et al. (2004); Raine et al. (2004)). If there is a TF interaction

with SRP, the nascent D1 - ribosome complex in Synechocystis sp. PCC 6803 (herein

also referred to as Synechocystis) would be a good system in which to examine it due to

D1 having a rapid turnover, a high abundance and naturally stalling during translation

(Tyystjarvi et al. (2001)). Hence, there are motivations for this research, namely, the

1

study of D1 integration into PSII, and the study of chaperone activities, which has

wider implications for the study of secretion and translation.

1.1 Photosystem II

1.1.1 The photosynthetic reaction

Photosynthesis is one of the most important physiological processes in the biosphere.

Almost all life on the planet depends either directly or indirectly on photosynthesis.

It is the process that converts light energy to sugars and also, importantly for animal

life, the production of O2 as a by-product. At the heart of photosynthesis is one of

the most chemically unfavourable reactions found in nature, the conversion of water

to protons and oxygen using light. This occurs in a series of reactions known as the

light reactions which can be broken into three different sections, the Z scheme, water

photolysis and oxygen photosynthesis. PSII, shown schematically in Figure 1.1; is the

structure at the centre of this reaction and is made up of at least 30 proteins (Yamamoto

(2001); Kamiya and Shen (2003)). The Z scheme is an electron transport chain that

starts with light energy being funneled into P680 by the Light Harvesting Complexes

that surround PSII. A negative charge is moved from the reactive TyrZ residue and

creates the charged P680* moiety. This charge then quickly propagates through to the

Pheophytin molecule, the primary quinone QA and the secondary quinone QB. The

charge removal from the TyrZ residue leads to the charge being removed from the

cluster of 4 manganese atoms which in turn removes the charge from water, donating

the electron and leaving a proton and molecular oxygen. The electron helps generate

the proton gradient to drive the generation of ATP and provides the electrons to

feed PSI and the generation of NADPH (Kamiya and Shen (2003)). The process of

the absorption of light and charge separation which leads to electron transport is the

subject of intense study, Figure 1.1 provides a broad outline of the process.

2

Figure 1.1: Photosystem II schematic. A photon is absorbed by P680, this increases its energy,

(P680*, not shown) very quickly transfers an electron to a pheophytin molecule (Pheo). This forms

a P680+Pheo− complex; the electron on the Pheo is then passed into the electron transport chain

via QA, QB and PQ then eventually to its ultimate destination of PS I and NADPH. On the lumenal

side of PSII, the positive charge migrates to the manganese centre. This accumulates four positive

charges, and catalyses the conversion of two H2O molecules to molecular O2 and H+ ions (Anderson

and Chow (2002)). Figure taken from Yamamoto (2001).

3

The D1 protein

If PSII is the heart of photosynthesis, D1 is the heart of PSII. It is a 471 amino

acid, 32 kDa protein and forms a heterodimer with the similar D2 protein (Yamamoto

(2001)). It has five transmembrane helices (Zhang and Aro (2002)) and its N-terminus

remains on the stromal side of the thylakoid membrane. It is encoded by the gene

known as psbA and is regulated at the level of translation in higher plants, but at

transcription in cyanobacteria (Zhang and Aro (2002)). However in both situations,

there is no translation of the complete protein in the dark even in the presence of ample

transcript located on the ribosomes (Zhang and Aro (2002)). Translation is initiated

and stalled at about 17 kDa in length. D1 synthesis in Synechocystis pauses at several

distinct sites between 17 and 25 kDa (Tyystjarvi et al. (2001)) only to go to completion

when associated with the PSII complex and in the presence of light.

1.1.2 Photoinhibition: The Damage and Repair cycle

Photoinhibition describes the situation which arises when the rate of photoinactivation

of PSII exceeds the rate of repair; this is usually due to an exposure of high light levels.

The primary cause of photoinhibition is the photoinactivation and destruction of the

D1 protein.

PSII catalyses very high-energy chemistry, the splitting of H2O, absorption of pho-

tons and charge separation. Hence, it is being constantly exposed to damaging oxy-

gen radicals and high-energy intermediates of the photosynthetic process. Of the two

proteins (D1 and D2) in the centre of the reaction, D1 is be the more readily dam-

aged (Baena-Gonzalez and Aro (2002)). The D1 protein helps bind the four catalytic

manganese ions, and is therefore more exposed to the reaction centre than D2. It

also contains the redox active tyrosine residue, TyrZ , and other molecules that are

highly reactive. The exposure of the protein to the Electron Transport Chain charged

molecules leads to the physical damage of the D1 protein. This directly causes photoin-

hibition when the flux of electrons thorough the complex and charged radicals created

therein, cause the degradation of D1 faster than the repair mechanisms can replace it.

When this damage occurs in plants, the PSII complex is transported form the grana

4

thylakoid stacks to the stroma thylakoid where the repair can take place (Barber and

Andersson (1992)). Shipton and Barber (1991) studied this effect in Peas in which the

damaged D1 protein it is first degraded and subsequently, fragments are released ).

The replacement of the D1 protein is essential to the continuation of photosynthesis

and the life of the plant or cyanobacteria.

1.2 Molecular Chaperones

1.2.1 Trigger Factor

Trigger Factor (TF) is an approximately 52 kDa molecular chaperone and has a pI

of about 5.2 (Moerschell et al. (1997)). In Escherichia coli it not only has chaperone

activity but also peptidyl-prolyl cis-trans isomerase (PPIase) activity (Valent et al.

(1995); Moerschell et al. (1997)). Trigger factor was initially discovered in 1987 as a

protein associated with ProOmpA (Crooke and Wickner (1987); Crooke et al. (1988))

and its translocation across the inner membrane. It was later discovered that it was

not necessary in vivo for this translocation (Guthrie and Wickner (1990)). Hence, the

focus of research on the protein has been on its PPIase functions and its chaperone

activity (Hesterkamp et al. (1996)).

PPIase activity was found in cellular fractions containing the 70S ribosome and the

50S ribosomal fragment. No PPIase activity was found in the 30S fragment. More

recently, trigger factor has been found to bind to the L23 subunit of the ribosomal

50S particle near the exit tunnel (Patzelt et al. (2001)). Not only this, but TF binds

promiscuously to nascent polypeptides as they emerge from the ribosomal exit tunnel,

with a preference for hydrophobic sequences (Patzelt et al. (2001)). TF binds between

60% and 100% of all ribosomes and exists in a 3:1 stoichiometry with the ribosome

(Crooke et al. (1988)). When bound to the ribosome it forms a relatively long lived

complex with an average binding time of about 30 seconds (Maier et al. (2003)). Given

the ubiquitousness of TF, the ability of it to bind almost all proteins and the relatively

long binding of TF to the ribosome, there are interesting questions to be asked about

its further interactions with other chaperone proteins. Trigger factor not only binds to

5

assist ribosomal processes, but to other molecular chaperones such as GroEL (Kandror

et al. (1995)). The GroEL/TF complex has a much-increased ability to bind to unfolded

proteins compared to just GroEL. Raine et al. (2004) found that there is simultaneous

binding of TF and the ribosome binding portions of the Signal Recognition Particle.

This interaction with several protein processing pathways indicates an important role

for TF in the protection of nascent chains and relocation to their cellular destination.

1.2.2 The Signal Recognition Particle

All cells need a method to move proteins into or across membranes. There are several

major ways of translocating proteins; the SecB pathway and the Signal Recognition

Particle pathway (SRP) are the independent main routes.

The SecB pathway targets completely translated proteins to a large membrane

structure which includes an ATP powered protein pump called SecA. It has three

associated proteins called SecY, SecE and SecG, as well as one of two associated protein

complexes (de Keyzer et al. (2003)). The SecYEG protein complex is collectively known

as the translocon.

The eukaryotic SRP is made of 7S RNA and six proteins (Bowers et al. (2003)).

The SRP in Streptococcus mutans (Gutierrez et al. (1999)) and E. coli is a complex

containing 4.5S RNA and the single protein Ffh (Fifty-four-homolog); it is recognised

by the receptor FtsY (Bowers et al. (2003)). The SRP interacts with nascent polypep-

tides by a GTPase subunit (Doudna and Batey (2004)). SRP binding stalls protein

translation, forming a complex which can bind to the receptor FtsY. Once the SRP-

FtsY-ribosome complex is formed at the membrane, the complex is transferred to the

translocon for insertion of the nascent peptide into the membrane.

This is similar to the process for proteins targeted by the SecB pathway indepen-

dently acting in parallel (Zhang et al. (2001)), see Figure 1.2 for a schematic diagram

of the pathway).

Recently Buskiewicz et al. (2004) showed that there was an interaction between

TF and the SRP. A requirement for the movement of a protein to its appropriate

location in the cell is the production of a signal sequence. It has been shown that

6

the sequence that the SRP binds and the sequence that TF binds are not mutually

exclusive ((Patzelt et al. (2001); Raine et al. (2004)). How TF and FFH compete for

binding to nascent polypeptides remains controversial.

1.3 E. coli experiments: What has been seen previ-

ously

Most of the data on TF comes from E. coli experiments. This is largely from character-

ising knockouts of the tig (TF) gene, biochemical characterisation of the trigger factor

protein and cross-linking studies. When ∆tig mutants were incubated at 4℃ they

showed about a 10-fold decrease in viability compared to WT (Kandror and Goldberg,

1997). By watching incorporation of radio labelled methionine when grown at 10℃,

the levels of TF were seen to increase two fold compared to WT. It was also shown that

the presence of HSPs (DnaK) had a deleterious effect on the cells at a low temperature.

TF knockouts also result in an increase in the number of nascent polypeptides that

associate with the heat shock protein DnaK. Cell morphology of E. coli TF mutants

was also seen to different from normal (Guthrie and Wickner (1990)). The normal size

of E. coli cells are 1 by 2 µm, the mutant cells were the normal diameter but very long

and filamentous. It has been proposed that TF is involved in cell division. The ∆tig

knockout showed that TF is not essential to the viability of the cells but causes modest

phenotypes.

1.4 Membrane Protein Integration

An alternative model suggests as well as acting separately, TF can cooperate in the

SecB pathway; this is shown in Figure 1.2. This model suggests that TF binds to other

export proteins such as SecB and DnaK (not shown in the graphic) (Lee and Bernstein

(2002)) and helps target some proteins to the translocon. For our example, TF may

bind the ribosome-D1 complex prior to the association with the thylakoid targeted

SRP, cpSRP54. TF is found to be bound to nearly all ribosomes, hence it is feasible

7

that TF binds the 17-25 kDa fragment of the D1 protein and stalls it at this point

until cpSRP54 can target it to SecY. Creating knockouts of most chaperones inhibits

the rate of protein export, the creation of a TF knockout increased the rate of export

(Lee and Bernstein (2002); Bowers et al. (2003)). This finding supports the idea that

TF stalls the production of the protein until it can be appropriately targeted, or that

it sequesters the protein slowing its production/release.

Figure 1.2: Two routes to the translocon. The SRP pathway to the left and the SecB pathway to

the right. Trigger factor is shown sequestering the protein prior to the integration or transport of the

nascent chain across the membrane. In the SRP pathway, binding of the SRP-nascent chain complex

to the receptor FtsY causes dissociation of TF (Buskiewicz et al. (2004)). It also shows the translocon

complex consisting of SecA and its associated proteins. Modified from de Keyzer et al. (2003).

1.5 Integration of D1 into the membrane

The translation of the D1 protein starts in the cytosol, then stalls until the SRP-D1-

ribosome complex is associated with the FtsY and PSII structure on the thylakoid

8

membrane (Tyystjarvi et al. (2001)). This initial product is a 17 kDa peptide (Baena-

Gonzalez and Aro (2002)) that includes two free transmembrane helices outside the

ribosome with one more helix in the 100A long ribosomal exit tunnel (Zhang et al.

(1999)). The translocation of the ribosome complex to the cpSecY-PSII complex re-

quires light. There is a weak interaction of the nascent-D1 ribosome complex with

the cpSecY, at the 17 kDa stage of the synthesis. Upon the light-dependent associa-

tion with cpSecY a fourth transmembrane helix is synthesised, auxiliary components

of PSII start to associate with the growing D1, and a disulphide bridge forms between

D1 and D2 (Zhang et al. (1999)). After this there is the rapid insertion of the 5th and

last transmembrane helix, post translational modification such as N and C terminal

processing, acetylation and phosphorylation (Sharma et al. (1997)), then the translo-

cation of the PSII complex back to the granal stacks of the thylakoid. This process is

depicted in Figure 1.3.

1.6 Synechocystis sp. PCC 6803 as a model

Synechocystis sp. PCC 6803 is a cyanobacteria that has the capacity to grow using both

photoautotrophic growth through photosynthesis and heterotrophic growth through

glycolysis of sugars. Due to the ability to grow on alternating energy sources it has

become a model organism for the study of photosynthesis and adaptability to other

plant stresses. Further to this, the photosynthetic processes found therein are very

closely related the processes found in plants. The whole genome sequence was released

in 1996 and has been reannotated several times since. Synechocystis readily undergoes

homologous recombination with plasmid DNA which allows mutants to be readily

made. These facts make Synechocystis a good model organism for studying disruptions

to genes potentially related to photosynthesis.

1.7 Research Hypothesis

Cyanobacteria are a well-characterised primary model for research into plant biochem-

istry. The laboratory strain Synechocystis can tolerate disturbance to its photosyn-

9

Figure 1.3: D1 replacement process. The D1 protein is damaged by exposure to high levels of light.

This activates the transport of PSII from the grana to the stroma. Here the complex is partially

disassembled, and the damaged D1 protein degraded. The SRP targets the ribosome to complete

translation of the new D1 protein directly into PSII. Upon the completion of this the complex re-

assembles and is transported back to the grana. Taken from Baena-Gonzalez and Aro (2002).

10

thetic abilities, as it is able to utilise glucose as an energy source. The insertion into

the membrane of the PSII protein D1 is a puzzle, the pieces of which are coming to-

gether. As the D1 protein is inserted using the SRP pathway, this system may provide

an experimental approach to investigating the SRP-TF.

Because TF binds nascent chains, especially hydrophobic ones (Patzelt et al. (2001))

D1 is a likely substrate. Prior to the association with the SRP the nascent chain of D1

may be bound to TF. It has been shown that TF and SRP bind in separate locations

on some polypeptides (Patzelt et al. (2001); Raine et al. (2004)). In my model, TF

interacts initially with the nascent D1 protein and then transfers the nascent chain to

the SRP chaperone-like moiety.

However, it seems likely that some nascent chains do not significantly interact with

TF. Some of these probably interact only with SRP. For some proteins, there are

multiple paths to their destinations. If there were a TF interaction with SRP and the

ribosome, the nascent D1 protein would be a good system in which to study it due

to its high abundance, predictable translation and rapid turnover. If TF is involved

in the process of D1 integration, the knockout may not have a dramatic change in its

photosynthetic activity is multiple redundant pathways exist. It is worth noting that

E. coli shows a limited phenotype for ∆tig or ∆dnaK mutants while the ∆tig∆dnaK

double mutant is lethal. However, as it is a protein that has a high throughput then

even a modest interruption in the pathway may be apparent. It is expected to display

a similar phenotype to E. coli in regards to cold sensitivity (Kandror and Goldberg

(1997)) and cell segregation (Guthrie and Wickner (1990)).

1.7.1 Research Aims

The aims of this research were to create a TFSpec mutant of Synechocystis sp. PCC

6803 which would then allow the characterisation of the phenotype of the Synechocystis

sp. PCC 6803 TFSpec mutant. If the knockout mutant shows a detectable phenotype,

an investigation into the function of the different functional domains of TF by creating

truncations of the protein could be done. Also a major point of interest is investigation

of the interaction of TF and FFH with the ribosome and via the D1 protein. Further to

11

this, using purified protein, biophysical characterisation of the Synechocystis sp. PCC

6803 TF protein can be attempted.

12

Chapter 2

Materials and Methods

2.1 General

Unless otherwise stated solutions were prepared with MilliQ water (MQ H2O) and

stored at room temperature.

2.2 Chemicals and Molecular Biology Reagents

Summarised in Table 2.1 are the chemicals and reagents used and their suppliers. All

other chemicals used were of analytical grade and from standard chemical suppliers.

Table 2.1: Chemicals

Chemical/Reagent Supplier

1 Kb DNA ladder Invitrogen Corporation, Carlsbad, CA, USA

30% Acrylamide/Bis solution Bio-Rad Laboratories, Hercules, CA, USA

β-Mercaptoethanol Sigma Chemical Company, St Louis, MO, USA

Agar Scientific Supplies, Auckland, NZ

Ammonium persulphate Bio-Rad Laboratories, Hercules, CA, USA

Bacto-Yeast extract Merck, Darmstadt, Germany

Bactotryptone Merck, Darmstadt, Germany

Bromophenol blue Peking Chemical Works, China

Complete, Mini, EDTA free (Cocktail Protease Inhibitor) Roche Diagnostics GmbH, Mannheim, Germany

Coomassie blue G-250 British Drug Houses, England

Crystal Screen HT kit Hampton Research, Alsio Viejo, CA, USA

Deoxynucleotide triphosphate Set Roche Diagnostics GmbH, Mannheim, Germany

Dithiothreitol Roche Diagnostics GmbH, Mannheim, Germany

continued on next page

13

Table 2.1: continued

Chemical/Reagent Supplier

Ethidium bromide Bio-Rad Laboratories, Hercules, CA, USA

Glass beads (100 µm) Sigma Chemical Company, St Louis, MO, USA

Glutathione sepahrose™ 4B Amersham Biosciences AB, Upsala, Sweden

Isopropyl β-D-thiogalactopyranoside Progen Biosciences, QLD, Australia

Kaleidoscope pre-stained protein standards Bio-Rad Laboratories, Hercules, CA, USA

Low-Mass SDS-PAGE marker Invitrogen Corporation, Carlsbad, CA, USA

Milk powder (Low Fat) Pams, Auckland, New Zealand

Sodium dodecyl dulphate British Drug Houses, England

TALON™ metal affinity resin CLONTECH Laborotaries, Palo Alto, CA, USA

NNN’N’- Tetramethylethylenediamine British Drug Houses, England

Molecular Biology Kits

Table 2.2 is a list of the commercially available kits that were used. Manufacturer’s

instructions were followed for the use of all molecular biology kits.

Table 2.2: Molecular Biology Kits

Kit Supplier

Gateway™ LR Clonase Enzyme Mix Invitrogen Corporation, Carlsbad, CA, USA

pGEM-T™ Vector System Promega, Madison, WI, USA

QIAquick™ Gel Extraction Kit QIAGEN®, GmbH, Germany

QIAquick™ PCR Purification Kit QIAGEN®, GmbH, Germany

QIAprep™ Spin Miniprep Kit QIAGEN®, GmbH, Germany

RadPrime DNA Labelling Kit Invitrogen Corporation, Carlsbad, CA, USA

Plasmids

Table 2.3 is a list of the commercially available plasmids that were used. Manufacturer’s

instructions were followed for their use.

Enzymes

Table 2.4 is a list of the enzymes used in the project and their suppliers. Manufacturer’s

instructions were followed for their use.

14

Table 2.3: Plasmids

Plasmid Supplier Feature

pGEM®-T Easy Promega Corporation, Madison, WI, USA EcoRV cleaved pGEM-5 vector with T overhangs

pGEM®-5 Promega Corporation, Madison, WI, USA Standard cloning vector

pENTR™11 Invitrogen Corporation, Carlsbad, CA, USA Entry vector for N-terminal tags

pDEST™15 Invitrogen Corporation, Carlsbad, CA, USA N- terminal GST tag

pDEST™17 Invitrogen Corporation, Carlsbad, CA, USA N- terminal 6x HIs tag

pET(gwA)-32 Gift of Robert Fagerlund, Wilbanks Lab N-terminal Thirodoxin and His tag

pUC19 Eaton-Rye Lab Standard cloning vector

Table 2.4: Enzymes used in this study

Enzyme Supplier

Taq DNA polymerase Roche Diagnostics GmbH, Mannheim, Germany

Expand High Fidelity PCR System Roche Diagnostics GmbH, Mannheim, Germany

Pwo DNA polymerase Roche Diagnostics GmbH, Mannheim, Germany

AvrI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

BamHI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

BglII restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

EcoRI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

EcoRV restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

NheI restriction endonuclease New England Biolabs, Beverly, MA, USA

SacI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

SalI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

SpeI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

SphI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

StuI I restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

PstI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

XbaI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

XhoI restriction endonuclease Roche Diagnostics GmbH, Mannheim, Germany

XmnI restriction endonuclease New England Biolabs, Beverly, MA, USA

XmaI restriction endonuclease New England Biolabs, Beverly, MA, USA

Poly nucleotide kinase Roche Diagnostics GmbH, Mannheim, Germany

Shrimp alkaline phosphatase (SAP) Boehringer Mannheim, Gmbh, Germany

T4 Polynucleotide Kinase Roche Diagnostics GmbH, Mannheim, Germany

T7 DNA ligase Roche Diagnostics GmbH, Mannheim, Germany

T4 DNA polymerase Roche Diagnostics GmbH, Mannheim, Germany

Klenow fragment DNA polymerase Boehringer Mannheim, GmbH, Germany

15

Specialty Equipment

Below in Table 2.5 is a list of the technical equipment used in this study.

Table 2.5: Specialty equiptment

Technical Equipment Source

Quantity One software Bio-Rad Laboratories, Hercules, CA, USA

GS-800 densitometer Bio-Rad Laboratories, Hercules, CA, USA

MasterCycler gradient Eppendorf AG, Barkhausenweg, Hamburg, Germany

100plus film developer All-Pro imaging, Hicksville, NY, USA

TL-100 UltraCentrifuge Beckman Coulter Inc., Fullerton, CA, USA

L-70 UltraCentrifuge Beckman Coulter Inc., Fullerton, CA, USA

Modified Erlenmeyer flasks Constructed by the University of Otago glass blower

Rank Brothers Oxygen electrode Rank Brothers Ltd, Cambridge, England

Hansatech Oxygen electrode Hansatech instruments, Norfolk, England

MacLab2e AD Instruments

Dynamic Light Scattering spectrometer Dynamics V.6, USA

DSM CD spectrophotometer OLIS, GA, USA

Mosquito Nanodrop Crystallisation Robot Molecular Dimensions Ltd, Suffolk, England

NanoDrop spectrophotometer Thermo Scientific Ltd, Wilmington, DE, USA

2.3 Cellular Manipulation

2.3.1 Growth and storage of E. coli and Synechocystis sp. PCC

6803 strains

E. coli strains were grown on (Luria-Bertani) LB plates or in LB broth as directed

including antibiotics as appropriate. Broth cultures were grown at 37℃ on a shaker at

170 rpm unless otherwise stated, plates were grown stationary at 37℃. Synechocystis

strains were grown at 30℃ under constant illumination of 25 µE m−2s−1 in aerated BG-

11 liquid media in modified Erlenmeyer flasks (Eaton-Rye, 2004) or on BG-11 plates

as appropriate. Antibiotics were used at the standard concentrations of Table 2.7,

unless otherwise stated. Once a new construct was created and verified, a culture was

grown to mid log phase in the appropriate antibiotics and a sample stored at -80℃ in

15% glycerol. Cyanobacterial strains were grown for 2-3 days in media containing

16

antibiotics; pelleted and resuspended in 2 mL BG-11/15% glycerol for storage at -

80℃.

2.3.2 Cell lines

In Table 2.6 is a list of the cell lines used in this project.

Table 2.6: Cell Lines

Cell Lines Source Antibiotic Genotype

Synechocystis sp. PCC 6803 Eaton-Rye lab stock None Glucose growth

Synechocystis sp. PCC 6803

TFSpec

Prepared in this study Spectinomycin Slow growth, decreased O2

evolution

E. coli pDEST™15 TF Prepared in this study Ampicillin Expression of GST tagged

TF

E. coli pET(gwA)-32 FFH Prepared in this study Ampicillin Expression of Thirodoxin

tagged FHH

E. coli DH5α ultra competent Wilbanks lab stock None Generic plasmid growth

strain

E. coli BL21 DE3 ultra com-

petent

Wilbanks lab stock None IPTG inducible expression

E. coli BL21 Star ultra com-

petent

Wilbanks lab stock None Disrupts RNase machinery

E. coli BL21 Rosetta 2 ultra

competent

Wilbanks lab stock None Complements rare tRNAs

E. coli pUC19 Spectinomycin Eaton-Rye lab stock Spectinomycin Spectinomycin resistance

2.3.3 Cyanobacterial Media

Below are tables that outline media for the growth of Synechocystis sp. PCC 6803.

BG-11 liquid media

Chemical Amount

100x BG-11 10 mL

1000x Ferric Ammonium Citrate 1 mL

1000x Na2CO3 1 mL

1000x K2HPO4 1 mLMade to 1 L with ddH2O.

17

BG-11 solid agar plates

Chemical Amount

1M TES/NaOH buffer pH 8.2 10 mL

Na-thiosulphate 3 g

Bacto-agar 15 gMade up to 1 L with BG-11 liquid media.

100x BG-11

Chemical Amount

NaNO3 149.6 g

MgSO4 · 7 H2O 7.49 g

CaCl2 · 2 H2O 3.60 g

Citric Acid 0.60 g

NaEDTA, pH 8.0, 0.25M 1.12 mL

Trace Minerals 100 mLMade up to 1 L with ddH2O.

Trace minerals

Chemical Amount

H3BO3 2.86 g

MnCl2 · 4 H2O 1.81 g

ZnSO4 · 7 H2O 0.222 g

Na2MoO4 · 2 H2O 0.39 g

CuSO4 · 5 H2O 0.079 g

Co(NO3)2 · 6 H2O 0.0494 gMade up to 1 L with ddH2O.

18

BG-11 minerals

The following are made up to 100 mL.

1000x Ferric Ammonium Citrate 0.6 g

1000x NaCO3 2 g

1000x K2HPO4 3.05 g

TES

Chemical Concentration

Tris, pH 8-8.5 50 mM

NaCl 50 mM

EDTA 5 mMMade up to 500 mL with ddH2O.

2.3.4 LB media

Chemical Amount

Bacto-tryptone 10 g

Bacto-yeast extract 5 g

NaCl 10 gMade up to 1 L with distilled water.

For plates 15 g agar were added per liter of media.

Prior to use, all media was autoclaved or filtered as appropriate.

19

2.3.5 Media supplements and their concentrations

All media supplements such as antibiotics, glucose and IPTG, unless stated otherwise,

were prepared with MQ H2O and filter-sterilised using a 0.22 µm Millipore filter. For

the incorporation into solid media, all supplements were added to sterilised agar which

had been cooled to less than 55℃.

Table 2.7 lists the different antibiotics used, the concentration that they were used

at and their supplier, unless stated otherwise. Ampicillin (Amp), spectinomycin (Spec)

and kanamycin (Kan) were prepared as stock solutions in MQ H2O. Chloramphenicol

(Chl) was prepared as a stock solutions in 100% ethanol. Antibiotics were stored at

4℃ or frozen at −20℃ if being stored for extended amounts of time.

Table 2.7: Media Supplements

Supplement Concentration Supplier

Ampicillin 25 µg.mL−1 Roche Diagnostics, GmbH, Mannheim, Germany

Chloramphenicol 34 µg.mL−1 United States Biochemical Corporation, USA

Kanamycin 50 µg.mL−1 Boehringer Mannheim, Gmbh, Germany

Spectinomycin 25 µg.mL−1 Sigma Chemical Company, St Louis, MO, USA

Glucose 5 mM Standard supplier

IPTG 1 mM Standard supplier

2.3.6 Transformation of competent E. coli cells

Transformants were created using ultra competent E. coli DH5α or BL21 DE3 cells,

which were prepared by others following the method described by Inoue et al. (1990).

The cells were thawed on ice slowly and 100 µL was gently pipetted into a pre-cooled

1.5 mL Eppendorf. Typically, 10 µL of the plasmid DNA (at approximately 300

ng.µL−1) was then added; this was then stored on ice for 30 minutes and subsequently

heat-shocked at 42℃ for 45 seconds. Tubes were then immediately cooled on ice for 2

minutes, 0.9 mL LB media added and incubated with shaking at 37℃ for 1 hour. Sub-

sequently, and 100 µL was plated onto LB plates containing the appropriate antibiotic

the remainder was centrifuged in a desktop micro-centrifuge for 1 minute, supernatant

20

removed and the cells were resuspended in 100 µL of fresh LB media then plated. The

plates were grown overnight at 37℃ and colonies selected for screening.

2.3.7 Cyanobacterial transformation

The transformation of Synechocystis sp. PCC. 6803 was done following the method of

Eaton-Rye (2004). All work was done in a laminar flow hood to prevent contamination.

A fresh 150 mL cyanobacterial culture was grown for 2-3 days in BG-11 and trans-

ferred from the flasks to sterile Falcon tubes. This was centrifuged at 4000 rpm at

room temperature in an IEC swinging bucket rotor, the supernatant removed and the

pellet resuspended in 2 mL BG-11 media. The optical density (O.D.) of a dilution of

the the cells was measured at 730 nm; for accuracy dilutions were done so the O.D.

was less than 0.4. For an effective transformation, the O.D. (A730) of the cells needs

to be 2.5. The dilution of cells required to achieve this O.D. is calculated by Equation

2.1 where 2.5(A730) is the desired O.D., F.vol(0.5 mL) is the desired final volume, O.D.

(A730) is the O.D. of the resuspended cells and Cell.Vol.Req is the volume of the cell

resuspension required to achieve the desired O.D. in the desired volume.

2.5(A730) ∗ F.vol(0.5mL)

O.D.(A730)= Cell.V ol.Req. (2.1)

The volume of required cells is then made up to a total volume of 0.5 mL with

an O.D. of 2.5 in sterile Eppendorf tubes, plasmid DNA (2-10 µL at approximately

300 ng.µL−1) ) is added to each tube and mixed gently. The tubes were placed in a

growth chamber at 37℃ for six hours with gentle mixing at three hours. Cells were

then spread on plates with the appropriate antibiotic and grown until colonies were

seen, these were re-streaked weekly to encourage segregation.

21

2.4 Cellular Studies

2.4.1 Photoautotrophic growth curves

Liquid cultures of Synechocystis sp. PCC. 6803 were grown until the O.D.730 was

between 0.8 and 1.0. The cells were centrifuged in sterile Falcon tubes at room tem-

perature for 10 minutes. The supernatant was removed, the pellet resuspended in 5 mL

of BG-11 media and then diluted to a volume of 40 mL. These steps were repeated

twice to remove remaining glucose from the original media and cells. After the last

wash the cells were resuspended in 2 mL BG-11 and the O.D.730 measured. The cells

were then diluted to give an O.D.730 of 0.05 in 150 mL. The flasks were then placed

in a growth chamber where they were aerated and grown under constant illumination

of 25 µEm−2s−1 at 30℃. Measurements were taken every 12 hours for the next 7 days

(168 hours). Prior to taking each reading, the flasks were topped up to 150 mL with

sterile MQ H2O. Density readings done were always measured below O.D.730 = 0.4 by

selecting appropriate dilutions during the course of the experiment.

2.4.2 Photoinhibition

Synechocystis sp. PCC 6803 cells were grown for several days in BG-11 media with

appropriate antibiotics and glucose at normal concentrations as specified in Table 2.7.

Once the cells reached log phase they were harvested as described in § 2.4.3 and made

to a chlorophyll concentration of 10 mg.mL−1. The cells, adjusted to the correct

concentration were then placed in the growth room in a 100 mL beaker on a stirring

block in front of a slide projector at such a distance that the cells were receiving

1000 µEm−2s−1 of light. The room temperature was maintained at 30℃ while the

O2 evolution was measured as per the method described in § 2.4.4, samples were taken

at t=0 and every 15 minutes. The projector was turned off at t=45 and hence the

light returned to the constant growth room light levels (25 µE m−2s−1) to allow the

recovery of the cells. This was done with WT cells and the TF Spec cells in completely

independent triplicates.

22

2.4.3 Chlorophyll estimation

Chlorophyll concentrations were calculated using the method determined by Arnon

(1949). Whole cells were resuspended in absolute methanol, chlorophyll was resus-

pended in 1.5 mL 80% v/v acetone, these were then vortexed and incubated on ice for

5 minutes. This was then centrifuged for 5 minutes at 12000 g and the A645 and A663

of the supernatant was measured. The concentration of chlorophyll was calculated by

the following equation:

TotalChl(µgChl.mL−1) = [20.2 ∗ A645] + [8.02 ∗ A663] ∗ dilutionfactor (2.2)

2.4.4 Oxygen electrode

Oxygen evolution measurements were made with a Hansatech DW1 O2 electrode unit,

a Hansatech LS2 light source control box CD1D. Data was recorded with a MacLab2e

(AD Instruments) and was controlled by Chart MacLab 3.6 software on a Macintosh

LCIII. The rate of oxygen evolution was measured by observing the change in oxygen

concentration in a suspension of Synechocystis sp. PCC. 6803 with a Clark-type oxy-

gen electrode. Light level was adjusted to 500 µmol.m−2.s−1 with a Melles Griot OG

590 sharp cut-off glass filter assisted by neutral density filters. Cells were resuspended

as described in § 2.4.3 and measured at 30℃ in BG-11 media. On insertion into the

electrode the cell resuspension was supplemented with the addition of 5 mM potassium

ferricyanide (FeCN) and 5 mM phenyl-1,4-benzoquinone (PBQ).

Formula 2.3 below shows the formula for the calculation of the O2 evolution rate of

Synechocystis sp. PCC. 6803 cells.

(XµmolO2.ml−1)(volume(mL))(60min.h−1)(∆mV.min−1)

(cal.constant(mV ))[Chl(mg)]= µmolesO2.(mg.Chl)

−1.h−1

(2.3)

23

Where the concentration of the dissolved O2 in water ‘X’ is 0.258 µ mmol O2.mL−1

at 30℃, ‘volume refers’ to the volume of cell resuspension in the reaction chamber, ‘∆

mV.min−1’ is the rate as measured, ‘cal.constant’ is the reading for the concentration of

dissolved O2 in air equilibrated water measured prior to the start of the measurements,

and ‘Chl’ is the amount of Chl in mg present in the reaction chamber.

2.5 DNA Isolation and Purification

2.5.1 Isolation of plasmid DNA

A single E. coli colony was taken from a plate that was incubated overnight at 37℃ and

inoculated into a 5 mL aliquot of LB as described in § 2.3.1. The overnight culture

was taken and pelleted in a micro-centrifuge at approximately 14,000g for 1 minute.

The Qiagen™ MiniPrep kit (see Table 2.2) protocol was followed for the rest of the

purification of the plasmid DNA.

2.5.2 Gel extraction of digested DNA

Restriction enzyme digested DNA was electrophoresed on a 0.8% agarose gel, as de-

scribed in § 2.6.1. The DNA band was visualised under UV light for a short period of

time and the band sliced from the gel using a sterile razor blade. The DNA from the

agarose gel fragment was purified using the QIAquick™ Gel Extraction kit according

to manufacturer’s specifications.

2.5.3 Chloroform/phenol extraction

Genomic DNA was isolated from strains of Synechocystis sp. PCC. 6803 following

Eaton-Rye (2004). Synechocystis was grown for three days in BG-11 media until dark

green. Falcon tubes were tared and 50 mL of cell culture per tube was centrifuged

for 5 minutes at 7000 rpm in a JA-17 rotor at 4℃. The supernatant was removed and

24

the weight of the pellet determined for future steps. The pellet was resuspended in

saturated NaI using 2 mL of saturated NaI for 1 g of paste. This was then incubated

at 37℃ for 20 minutes. After this the NaI was diluted by filling the tube with room

temperature sterile MQ H2O, then centrifuged at 4℃ for 10 minutes at 4000 rpm in the

IEC refrigerated centrifuge and the supernatant removed. The pellet was resuspended

in 8 mL of TES per gram of cells this was done by pipetting through a 1 mL disposable

pipette tip. Per gram of cells, 75 µg of lysozyme (using a 50 mg.mL−1 stock) in H2O

was mixed gently and incubated at 37℃ for 20 minutes.

After incubation 1 mL N-lauryl sarcosine per gram of cells was added, mixed care-

fully and incubated at 37℃ for 20 minutes. The lysate was put into a round bottomed,

screw-capped plastic tube and an equal volume of equilibrated phenol added to each

tube. Parafilm was wrapped around the lid to prevent leakage and the solution was

mixed gently on a wheel for one hour. This was then centrifuged for 10 minutes at 4000

rpm in an IEC refrigerated centrifuge and the pink aqueous upper layer was recovered

using a P5000 pipette. An equal volume of chloroform was added to the aqueous phase,

and this was mixed on the wheel for 45 minutes with parafilm around the lid to prevent

leakage. This was then centrifuged for 10 minutes in the IEC refrigerated centrifuge

and the top phase transferred to a tube compatible with the JA-17 rotor. Next, 1/10th

volume of 3M Na-acetate (pH 5) and 2 to 2.5 volumes of 100% ethanol was added,

inverted and stored overnight at -20℃.

This was centrifuged for 10 minutes at 14000 rpm in the JA-17 rotor and the ethanol

removed. It was then washed again with cold ethanol, centrifuged for 5 minutes at 4000

rpm in the IEC refrigerated centrifuge, and most of the ethanol aspirated. Finally, the

pellet was dried and subsequently resuspended in 300 µL TE. The DNA preparation

was then stored at -20℃.

25

2.5.4 Ethanol precipitation

DNA to be precipitated was deposited in a micro-centrifuge tube and 1/10th volume

solution 3M CH3COONa pH 5.2 and 2.5 volumes of ice cold 100% ethanol were added.

The solution was kept at -80℃ for at least 30 minutes. The DNA was subsequently

spun at 4℃ in a desktop micro-centrifuge at approximately 14,000g for 15 minutes.

The supernatant fraction was discarded the pellet washed with 70% v/v ethanol in

water and spun at approximately 14,000g for 15 minutes. The DNA pellet was then

dried in a 37℃ heat block until the all the ethanol had evaporated and resuspended in

an appropriate volume of Qiagen buffer EB.

2.5.5 Polymerase chain reaction

Polymerase Chain Reaction (PCR) was carried out using a standard protocol that was

then varied to search for the optimal conditions. The standard protocol that was used

is illustrated in Table 2.8 and performed in an Eppendorf MasterCycler Gradient. The

standard recipe for PCR is outlined in Tables 2.9 and 2.10, which were mixed immedi-

ately prior the start of the cycling reaction. Paraffin oil was applied to the surface of

the reaction mix to prevent evaporation.

2.5.6 Purification of PCR products

PCR was done as described in § 2.5.5 and captions of figures that contain a PCR

product. PCR purification was done using a QIAquick™ PCR purification kit (See

Table 2.2) according to the manufacture’s instructions.

Colony PCR

Colony PCR was done using a normal PCR protocol but substituting a small volume

of cells for template DNA. This was then mixed using a sterile pipette tip. The correct

amount of cells acting as the template was determined by the final colour of the PCR

26

mixture. This, when done with was a pale glass green. The primers used for the analysis

were the same as used for the initial amplification of the gene (TF interruption forward

and TF interruption reverse, Table 2.11). Several replicates of each of the samples were

always run due to variable success of the PCR reaction.

Table 2.8: PCR template protocol

Step Temperature Time

Denaturing 94℃ 5 minutes

Denaturing 94℃ 20 seconds

Annealing 55 ± 5℃ 30 seconds

Extension 72℃ 40 seconds

Repeat 30 times

Extension 72℃ 5 minutes

Hold 4℃ indefinitely

Table 2.9: PCR Mix 1

Mix 1 Volume or concentration

Forward primer 0.4 µM

Reverse Primer 0.4 µM

Template DNA 0.3 (approx. 20 ng)

dNTP mix 0.4 µL

Sterile MQ H2O 8.5 µL

Total 10

dNTP mix was made in accordance with the manufacture’s instructions.

2.5.7 Primers for Polymerase Chain Reaction

The primers in Table 2.11 for the PCR reactions were synthesised by Invitrogen, NZ.

Primers T7 and SP6 were supplied by Alan Wilson Centre Genome Service (AWCGS).

The primers for the sequencing of pENTR-11 were synthesised by Invitrogen and sup-

plied by Robert Fagerlund. Truncation primers are labelled A-h, forward primers are

denoted with capital letters and the reverse direction primers are in lowercase lettered

primers.

27

Table 2.10: PCR Mix 2

Mix 2 Volume (µL)

Expand 10x Buffer 2

Expand Polymerase 0.15

Sterile MQH2O 7.85

Mg2+ 0

Total 10

Table 2.11: Primers

Primer Name Sequence 5’ to 3’ (Feature underlined) Feature

TF Interruption forward GGC AAA GCT TAC TCC TCC AG N/A

TF Interruption reverse CGG TAT GCC TCT GAC ACC CC N/A

TF GateWay forward CTG GAA GTT CTG TTC CAG GGG CCC ATG AAA

GTT ACC CAG GAA AAA TTG CCA GAT

3C protease site sequence

TF GateWay reverse GGC CCT CGA GTT ACT CTT CGT CGG A XhoI site

FFH GateWay forward CTG GAA GTT CTG TTC CAG GGG CCC ATG TTT

GAT GCC TTA GCC GAT CGC CTT GAA

3C protease site sequence

FFH GateWay reverse GGC AAA TCT CGA GTT AAA GCT GGC CAA AAC

C

XhoI site

TF truncation A CTA ATC GGG GCA TGC AGA TGG GCA SphI site

TF truncation B TTC CCA TCA ATG AAG ATA TCC CAG GAA EcoRV site

TF truncation c TTC CTG GGA TAT CTT CCT CAT TGA TG EcoRV site

TF truncation d GCT TCG AGG CCT ACA TCC GCT G StuI site

TF truncation e CCG GGA CAA CTA GTG CCC GTT T SpeI site

TF truncation f CGC TGT TTG GCT AGC TCC TGG AAT NheI site

TF truncation g GGG TTA CTC TAG ATC GGA GGT TG XbaI site

TF truncation G CAA CCT CCG ATC TAG AGT AAC CC XbaI site

TF truncation h GTT AGG TCG ACG GAA AGG TAG GGA AAA T SalI site

Chl (EcoRV) fwd CGA ATA GAT ATC TGT GAC GG EcoRV site

Chl (XbaI) rev AAT TTC TAG AAT TCA TCC GC XbaI site

T7 TAA TAC GAC TCA CTA TAG GG Sequencing primer

SP6 ATT TAG GTG ACA CTA TAG Sequencing primer

28

2.5.8 Restriction endonuclease digestion

Restriction digests were done according to the manufacturer’s instructions with consid-

eration given to available buffers, temperature of the incubation, concentration of DNA

and time desired to achieve the complete digest. Typically between 200 and 500 ng of

DNA were digested in a mix that was optimised for minimum volume. Double digests

were done as required in restriction buffers and incubation temperatures that were

compatible for both of the enzymes used. If that was not possible, DNA was digested

with one enzyme, purified with the QiaQuick PCR purification kit or EtOH precipita-

tion (see § 2.5.6 and 2.5.4), and then digested with the next enzyme. In one instance

where buffers differed only in the concentration of some components, the buffer was

modified by addition of the components required to make the two buffers equivalent

the conversion buffer is outlined in the table below.

Roche SuRE buffer M to H conversion buffer

Chemical Amount

Tris HCl, pH 7.5 40 mM

NaCl 50 mM1 µL was added to every 10 µL of M buffer digestion.

2.5.9 Ligation

Ligations were done using vector and insert DNA digested as per §2.5.8 and purified by

QIAquick gel extraction or QIAquick PCR purification as described in §2.5.6 or §2.5.2.

DNA was combined at a ratio of approximately 1:3 (vector:insert), with 50 ng of vector

used. Addition of 2 µL of 10x ligation buffer, 1 µL of T4 DNA ligase (1U) and sterile

MQ H2O gave a final volume of 20 µL. Ligations were incubated at 22℃ overnight.

Typically, 10 µL of the ligation was used in the subsequent transformation.

29

2.6 DNA Analysis

2.6.1 Agarose gel

Agarose gels were made in 1x TAE buffer, with 0.8% (w/v) agarose. The agarose was

dissolved by heating in a microwave oven, poured, and set in a BioRad gel caster with

8 or 15 well combs. The gel was then laid in an electrophoresis tank with 1x TAE

buffer covering the entire gel. DNA with loading solution was loaded into the wells via

pipette. Typically, the marker (Invitrogen 1kb ladder) was loaded in the first well and

DNA was electrophoresed for 40 minutes at 90V.

For staining, ethidium bromide (EtBr) was added into TAE buffer a final concentra-

tion of 0.25 mg.L−1. After running the gel it was stained for 20 minutes on a shaker at

15 rpm. DNA bands were visualised by fluorescence under UV light, and photographed

using the Bio-Rad Eagle-Eye Gel Dock using Quantity-One software.

50x Tris-acetate-EDTA (TAE) buffer


Tris-HCl (pH 8.0) 2 M

EDTA 50 mMAdjusted to pH 8.3 with glacial acetic acid.

6x DNA loading solution

Chemical Amount

Glycerol 30% (v/v)

Bromophenol blue 0.25% (w/v)

Xylene cyanol 0.25% (w/v

30

1kb DNA ladder

Chemical Amount

1kB DNA ladder 10 µL

DNA loading solution 20 µLMade to 100 µL with MQH2O.

2.6.2 Quantification of DNA and proteins

DNA was quantified by measuring absorbance (A260 and A280) using the Beer-Lambert

Law as measured and calculated on a NanoDrop Spectrophotometer. DNA concentra-

tion and an indication of purity was computed automatically by the software comparing

the A260/A280 ratio. Typically, a 1.5 µL sample was used.

2.6.3 DNA sequencing

DNA was adjusted to the requested concentration for sequencing by the Allan Wilson

Centre Genome Service (AWCGS) based at either the Massey or Albany campus on a

ABI 3730 Genetic Analyzer. If necessary, primers were added to the appropriate con-

centration; otherwise, T7 and SP6 primers were supplied by the AWCGS. Sequencing

results were analysed using 4Peaks Version 1.71, Sequencher Demo version 4.5, and

Enzyme X version 3.

2.6.4 GateWay recombination

GateWay recombination was done as directed by the manufacturer.

2.7 Southern Blot Procedure

2.7.1 Southern transfer blot

Genomic DNA (5 mg) was completely digested as per § 2.5.8 with appropriate restric-

tion enzymes and run on an agarose gel as in § 2.6.1 in a gel caster containing a

31

fluorescent ruler. An empty lane was left between the marker and the digested ge-

nomic DNA. This was then photographed in the Eagle Eye UV imager with the gel

still in the caster so the fluorescent ruler was visible. The gel was trimmed to the

size required and the bottom right corner removed for orientation purposes. This was

then soaked in 0.25 M HCl for 10 minutes, rinsed in water, and subsequently soaked

in 0.4M NaOH for 30 minutes. A piece of GeneScreen™ membrane and three pieces of

Whatman 3MM (Whatman) paper was cut to the size of the gel and labelled using a

pencil. The membrane was rinsed in distilled water for 2 minutes and in 0.4 M NaOH

again for a further 10 minutes. A capillary blot tray was set up with two reservoirs of

0.4 M NaOH with a Whatman paper wick lying over a bridge with both ends in the

reservoir. The gel was placed on top of the wick, with the membrane and the three

sheets of Whatman paper layered on top. Stacks of paper towels were placed on top of

the Whatman paper and held down using strips of masking tape. The gel/membrane

capillary apparatus was left overnight to transfer the DNA to the membrane. After

the transfer time, the apparatus was disassembled and the membrane kept damp for

further processing.

2.7.2 Probe creation

This is a modification of the method outlined in the Invitrogen RadPrime DNA La-

belling System. A fragment of genomic DNA was prepared either by PCR (§ 2.5.5) or

restriction digest (§ 2.5.8). Denatured DNA was created by dissolving 25 ng of 5-20 µL

in sterile MQ H2O, heated to 100℃, then cooled immediately on ice. The [α 32P]dCTP

labelling mix was added, mixed briefly, then 0.5 µL of Klenow Fragment was mixed in

gently but thoroughly. The mix was then centrifuged briefly at approximately 14,000g,

incubated at 37℃ for 10 minutes, 2.5 µL of Stop Buffer added, and then the total

reaction was spun through a glass wool filter for 1 minute into a tube containing 1 µL

of 10 mg.mL−1 salmon sperm DNA. The probe was then denatured again for 10 min-

utes, stored on ice for 5 minutes, added to the membrane then incubated overnight at

37℃ while mixing on a rotator.

32

2.7.3 Pre-hybridisation and probing

After the overnight incubation, the gel was checked for any bands that were not trans-

ferred to the membrane by staining the gel in EtBr (§ 2.6.1). The membrane was then

immersed in 2x (Standard Sodium Citrate) SSC for 2 minutes and dried under a lamp

for 30 minutes. The membrane was placed into a Pre-hyb bottle with 10 mL of Den-

hardts Pre-hybridisation Solution and rotated at 37℃ for 6 hours. The radio-labelled

probe was added to the bottle containing the membrane and 10 mL pre-hybridisation

solution and incubated at 37℃ overnight. After the overnight binding, the membrane

was washed twice in 2x SSC for 5 minutes at room temperature and then twice for 30

minutes in 2x SSC with 0.2% SDS. This was followed by two washes in 0.1x SSC at

room temperature for 10 minutes. The membrane was then enclosed in plastic wrap

and placed in a film cassette with Kodak BioMax XAR film for two days at -80℃.

2.7.4 Film development

The membrane to be developed was encased in plastic wrap and taped securely in

place within the cassette. In a dark room with red light illumination, film marked

for orientation determination after development was placed over the membrane. The

cassette was securely closed and a timer was started to measure the elapsed time of

exposure of the film. After the desired exposure, the film was removed and developed

in the Kodak Automatic film development machine. Once the exposure levels were

examined, it was determined whether more or less further exposure was required and

the process repeated.

100x Denhardts Solution

Chemical Amount

Ficoll 400 1 g

Polyvynlpyrrolidone 1 g

BSA (Fraction V) 1 gMade up to 50 mL with MQ H2O, filter sterilised and stored at -20℃.

33

20x SSC

Chemical Amount

NaCl 175.3 g

Na3Citerate ·H2O 88.2 g

H2O 800 mLAdjusted to pH 7.0 with 1 M HCl and made to 1 L with MQ H2O

Denhardts Pre-hyb Solution

Chemical Amount

Denhardts solution 5x

SSC 5x

Formamide 50%

SDS 1%Made to volume with dd H2O.

[α-32P]dCTP labelling mix

Chemical Amount

dATP 0.5 µL of 500 µM

dGTP 0.5 µL of 500 µM

dTTP 0.5 µL of 500 µM

[α-32P]dCTP 2 µL of 3000 Ci/mmol, 10 mCi/ml

Random Primers Solution 10 µL of 2.5x solutionMade to 25 µL with sterile MQ H2O.

34

2.8 Protein Expression

2.8.1 Induction of lac promoter constructs

Expression of recombinant proteins was done using the following method, unless oth-

erwise stated. A single E. coli colony from a LB agarose plate was inoculated into

5 mL of LB containing appropriate antibiotics and grown overnight on a shaker at

37℃. A 50 µL sample of the cells was inoculated again into 5 mL of LB and grown

to mid log phase (O.D.600 ≈ 0.6). This 5 mL culture was then inoculated into a 2 L

baffled Erlenmeyer flask with 400 mL of LB containing appropriate antibiotics. Cul-

tures were grown at 37℃ until they reached an O.D.600 of between 0.3 and 0.6. A

1 mL (uninduced) sample was removed and IPTG added to the remaining culture to

a final concentration of 0.5 mM, before incubation for a further 3 hours at 37℃. Cells

were then harvested though two steps of centrifugation at 10,000 rpm for 15 minutes

(Beckman JA-14 rotor, 4℃). Cell pellets were stored at -20℃.

2.8.2 Lysis of cells

Frozen cell pellets were resuspended in PBS so a 1:50 dilution of the resuspension had

an O.D.600 of 1. At all stages following resuspension, samples were stored on wet ice

and in 1 mM PMSF. Cells were lysed by 15 cycles of 30 s sonication with a large probe

and set at 40% (Sonifier Cell Disruptor, Branson Sonic Power Co.) separated by 30 s

intervals until the 1:50 dilution of the resuspension had an O.D.600 of 0.1. Lysate was

separated into soluble and insoluble fractions by two steps of centrifugation at 12000

rpm for 30 minutes (Beckman JA-17 rotor, 4℃).

35

Phosphate Buffered Saline (PBS)


NaCl 140 mM

KCl 2.7 mM

Na2HPO4 10 mM

KH2PO4 1.8 mMMade up to 1 L with MQ H2O, pH 7.3.

2.8.3 GST resin

GST resin purification was done using the batch method. The resin was washed three

or more times with PBS, each time using approximately 10 resin volumes prior to the

use of the resin, centrifuging at 500 g for 5 minutes between steps. The clarified lysate

was then incubated with washed resin on a shaker at 4℃ for 1 hour or longer. The resin

was centrifuged for 5 minutes at 500 g and washed 3 times with PBS, each time using

approximately 10 resin volumes, centrifuging at 500 g for 5 minutes between steps. The

protein was released from the resin by 3C cleavage at the 3C protease site engineered

between the GST tag and the recombinant protein. Cleavage was carried out in 5 mM

DTT for 72 hours at 4℃ and the protein quantified by NanoDrop spectrophotometry.

2.8.4 Talon resin

Talon resin purification was done using the batch method. The resin was washed three

or more times with PBS, each time using approximately 10 resin volumes prior to the

use of the resin. The clarified lysate was added to the resin and mixed on a shaker at

4℃ for 1 hour or longer and the resin pelleted by centrifugation at 500 g for 5 minutes.

The resin was then washed three times with PBS, each time using approximately 10

resin volumes, centrifuging at 500g for 5 minutes between steps. Protein was then

eluted from the resin with three sequential elutions of 1.5 mL 500 mM imidazole in

PBS pH 7.3 and one final elution of 1.5 mL PBS. These fractions were pooled and

protein was released from the tag by 3C cleavage at the 3C protease site engineered

36

between the thirodoxin tag and the recombinant protein. Cleavage was carried out

in 5 mM DTT for 96 hours at 4℃. This was then dialysed three times for 4 hours

in 1 L of PBS to remove DTT and imidazole. The dialysed protein was then bound

back to to the resin for 1 hour to remove the thirodoxin tag and other proteins that

bind specifically to the resin. This was then centrifuged for 5 minutes at 500 g. The

supernatant containing the desired protein was removed and the resin washed with 2

mL PBS, then centrifuged for 5 minutes at 500 g to recover any residual aggregated

protein.

2.8.5 Size-exclusion chromatography

Size-exclusion chromatography (SEC) was performed using a HiLoad™ 200 Superdex™ col-

umn at 1 mL.min−1. The column was rinsed with at least two column volumes of

MQ H2O and then equilibrated with at least two column volumes of running buffer

prior to sample loading. Samples were loaded in the smallest practical volume, with

maximum load volumes of 5 mL.

2.8.6 Protein concentration

Protein concentration was done using a Centricon Spin Purification column in accor-

dance with the manufacturer’s instructions. Samples of the flowthrough not containing

the protein of interest were kept for analysis.

2.8.7 Ribosome and associated proteins preparation and anal-

ysis

Ribosome isolation

The ribosome extraction below is a combination of a mini-thylakoid preparation from

Eaton-Rye (2004) for the cell lysis and a polysome prep (Tyystjarvi et al., 2001) for

ribosome sedimentation.

37

Cells were grown in BG-11 media for several days until they were mid to dark

green. Initially the cells were pelleted in a GSA rotor at 4000 g for 7 minutes. The

pellet was then washed with 5 mL of Washing buffer, pelleted again, washed with 5 mL

of Isolation buffer, pelleted again, and then washed with 5 mL of Isolation buffer with

RNAse inhibitor and a cocktail of protease inhibitors. A half-volume of 150-212 µm

beads were added and vortexed 6 times for 1 minutes for cell lysis. Isolation buffer

without sucrose was added and centrifuged at 650 g for 5 minutes. The supernatant

fraction was then removed and centrifuged for 20 minutes at 18,000 g to separate the

membrane and the cytosolic fractions. The membrane pellet was resuspended in 500

µL isolation buffer without sucrose and containing 2% v/v polyoxyethelene-10-tridecyl

ether. For the supernatant fraction, polyoxyethelene-10-tridecyl ether was added to

2% v/v. Fractions were then incubated on ice for 10 minutes then centrifuged for 5

minutes at 18000 g. The supernatant of each of these fractions was layered on a sucrose

cushion, then centrifuged in a micro-ultracentrifuge for 1 hour at 270,000 g to pellet

the polysomes.

Special care was taken with the contents of the preparation as the centrifuge tubes

are sensitive to some solvents. Also additional care was taken to ensure no glass cell

lysing beads were carried over for the fast spin. The tubes were balanced to within

0.001 grams of each other, the limitation of the scales that were to be used.

2.8.8 Ribosome Preparation

HMCS Solution


Hepes/NaOH pH 7.2 50 mM

MgCl2 10 mM

CaCl 5 mM

Sucrose 1 M

38

Washing Buffer


Sucrose 400 mM

Tris pH 8.5 50 mM

MgCl2 10 mM

EDTA 30 mM

Chloramphenicol 0.5 mg.mL−1

Isolation Buffer


Sucrose 400 mM

Tris pH 8.5 50 mM

MgCl2 10 mM

Chloramphenicol 0.5 mg.mL−1

2.9 Protein Analysis Techniques

2.9.1 Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophore-

sis

Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis (SDS-PAGE) was car-

ried out in accordance with the method by Bollag et al. (1996). The samples were run

on a 12% acrylamide gel with a 5% stacking gel made using the Bio-Rad Protean™ 3

system. Samples were loaded with a 2:1 ratio of sample to loading mix to a total

volume of 15 µL. The gels were run at 100 V through the stacking gel and then 200 V

through the resolving gel until the dye front exited the bottom of the gel. Low Range

SDS-PAGE Standard or Kaleidoscope® was used as a marker in accordance with the

manufacturer’s instructions. The gel was then either stained with Coomassie (stained

for 30 minutes and then destained overnight) or electro-transferred to membrane.

39

SDS-PAGE Buffers (Bollag et al. (1996))

4x Resolving Gel Buffer

Chemical Amount

Tris-base 36.34 g

MQ H2O 160 mL

20% SDS 4 mLMade up to 200 mL after adjusting pH to 8.8 with HCl.

4x Stacking Buffer

Chemical Amount

Tris-base 12.12 g

MQ H2O 160 mL

SDS 20% 4 mLMade up to 200 mL after adjusting pH to 6.8 with HCl.

Recipe for two Protean® 3 gels:

Resolving Gel 12% (10 mL)

Chemical Amount

30% Acrylamide stock 4.0 mL

4x Resolving Buffer 2.5 mL

MQ H2O 3.5 mL

APS (0.1g.mL−1) 120 µL

TEMED 10 µL

40

Stacking Gel 5% (5 mL)

Chemical Amount

30% Acrylamide stock 0.83 mL

4x Stacking Buffer 1.25 mL

MQ H2O 2.92 mL

APS (0.1g.mL−1) 25 µL

TEMED 8 µL

10x Running buffer Stock solution

Chemical Amount

Glycine 144 g

SDS 10 g

Tris 30 gMade to 2 L with ddH2O.

Lysis Buffer 100 mL

Chemical Amount

SDS 3g (3% w/v)

Tris-HCl 0.757 g

Glycerol 10 mLpH was adjusted to 6.8 with concentrated HCl.

3x Sample Loading Buffer

Chemical Amount

Lysis Buffer 0.5 mL

Bromophenol Blue 40 µL

β-Mercaptoethanol 25 µL

41

Stain (500 mL)

Chemical Amount

Coomassie G-250 0.1%

Iso-propanol 125 mL

Glacial Acetic Acid 50 mLMade to 500 mL with H2O.

Destain

Chemical Amount

Iso-propanol 50 mL

Glacial Acetic Acid 70 mL

Glycerol 40 mLMade to 1 L with H2O.

2.9.2 Electro-transfers

For Western Blot analysis the SDS-PAGE gel was blotted to a nitrocellulose membrane

in the Bio-Rad Criterion blotter according to manufacturer’s instructions. The stacker

gel was removed and one corner was cut for recognition before the gel was placed on

two layers of damp Whatman 3MM blotting paper. The nitrocellulose was layered on

top and a roller used to remove air bubbles, then two more layers of blotting paper

were placed on top. Transfer was preformed in a 4℃ room at 100 V for 30 minutes

with Transfer Buffer to fill line, a magnetic stirrer and an ice pack. After transfer,

the acrylamide gels were stained with coomassie to ascertain weather the transfer had

occurred.

2.9.3 Western blots

The nitrocellulose was blocked in 5% milk powder in TBS-T for an hour, then rinsed

in TBS-T for 15 minutes and three times for 5 minutes. The primary antibody (a

42

polyclonal antibody raised in rabbits) was applied overnight in concentrations indicated

in Table 2.12 and then rinsed three times with TBS-T, once for 15 minutes and three

times for 5 minutes. The Goat Anti-Rabbit HRP secondary antibody was applied for

an hour at the concentration indicated in Table 2.12. This was then rinsed once for

15 minutes and four times for 5 minutes each. The membranes were then incubated

in the dark room for 1 minute with the ECL reagent mixed at a 1:1 ratio of Solution

A:Solution B. The membrane was then placed between two layers of plastic wrap.

Kodak BioMax XAR radiography film was exposed to the blot for between 10 seconds

and 15 minutes.

Transfer Buffer


Tris-base 20 mM

Glycine 150 mM

Methanol 20% (v/v)

SDS 0.05% (w/v)

2.9.4 Western Recipes

TBS-T


Tris 50 mM

NaCl 150 mM

Tween 20 0.1% (v/v)pH adjusted to 7.5.

43

Transfer Buffer


Tris 50 mM

Glycine 150 mM

SDS 0.05% (w/v)

Methanol 20% (v/v)Made to volume with ddH2O.

2.9.5 ECL reagents

Solution A


Tris-HCl pH 8.8 100 mM

Luminol 10 µM

p-Coumaric Acid 0.526 µM

Solution B (dark stored)


Tris-HCl pH 8.8 100 mM

H2O2 30% (v/v)

2.9.6 Antibodies used in Western Experiments

Below in Table 2.12 are the antibodies used in the western blot experiments and their

dilutions.

N-terminal sequencing

A salt-free protein sample was provided to the Protein Microchemistry Facility (PMF)

by using a Prosorb® sample preparation cartridge. The cartridge was prepared by

44

Table 2.12: Antibodies

Antibodies Dilution Source

Goat Anti-Rabbit immunoglobulin s/HRP 1:17,000 DakoCytomation A/C,

Glostrup, Denmark

Rabbit Anti-TF raised against Aquifex aeolicus 1:5,000 Gift of Stu McInnes,

Wilbanks lab

Rabbit Anti-TF raised against Heamophilus influenzae 1:10,000 Gift of Esther Pearl,

Wilbanks lab

Rabbit Anti-TF raised against Escherichia coli 1:5,000 Gift of B. Bukau, Zentrum fur

Molekulare Biologie, Univer-

sitat Heidelberg

Rabbit Anti-TF raised against Synechocyctis sp. PCC. 6803 1:100,000 Generated in this study

Rabbit Anti-FFH raised against Synechocystis sp. PCC. 6803 1:100,000 Generated in this study

Rabbit Anti-TF raised against Mycobacterium tuberculosis 1:5,000 Raised by Dr S.J. Clark,

Wilbanks lab

pre-wetting with 10 µL of 100% methanol. This was followed by 100 µL of 0.1%

Trifluroacetic acid (TFA). Approximately 3 µg of protein was added to 100 µL 0.1%

TFA and drawn through the membrane by capillary action into an absorbent pad.

The membrane was then washed with a further 100 µL of 0.1% TFA and given to the

PMF for further processing and sequencing on an Applied Biosystems 490A Gas Phase

Protein Sequencer.

2.10 Antibody Creation and Testing

2.10.1 Ethics approval

Ethics approval was sought from the Otago University Ethics Committee which weighed

up the research benefits against the harm to society and the rabbits. Permission was

subsequently granted to immunise four white rabbits, two rabbits for each protein.

This was to increase the chances of obtaining a good titer and immunogenic response.

45

2.10.2 Immunisation Protocol

Protein was purified as previously described and mixed with Freund’s Complete Adu-

jvanct for the initial immunisation, then administered by a veterinary technician at

the Hercus Taire Research Unit. Prior to immunisation, an initial test bleed was done

which allowed an examination of the test bleed for initial infection and immunity to

proteins that could be assayed for. Subsequent injections containing Freund’s Incom-

plete Adjuvant were administered with the same method. Test bleeds were provided

at three and six weeks. Once the rabbits had been immunised with the third booster

and an appropriate period of time waited for the effect of this booster to have taken

place, it was determined that the immunity would not develop any more against the

presented antigen. Hence, the rabbits were further test bled and subsequently exsan-

guinated. The serum was collected and divided into manageable aliquots and stored

for future use.

2.10.3 Dot-blots

Dot blots were carried out to examine a range of serum concentrations and using

appropriate controls. Attempts were made to quantify the amount of protein in cell

lysates; however assays on cell lysates using standard assay procedures were found to

be inaccurate.

2.11 Biophysical Characterisation

2.11.1 Dynamic light scattering

Dynamic light scattering (DLS )was done using the Dynamics V.6 machine and associ-

ated software. The blank was set using a freshly cleaned and dried cuvette filled with

PBS buffer. Readings were collected as 10 acquisitions using protein in PBS. Data

analysis was done by Dynamic V.6 software.

46

2.11.2 Circular dichroism

Pure protein samples, as analyzed by DLS were taken and clarified by centrifugation

at approximately 14,000g at 4℃ to ensure an absence of particulate matter in the

sample. The samples were then stored on ice until insertion into the cylindrical cuvette

for analysis in the OLIS DSM CD spectrophotometer. The baseline was run on PBS

buffer and set as the reference.

Single temperature scan

Baseline and sample spectra were collected from 190 nm to 260 nm in 2 nm increments.

This data range was input into a graphing program which determined data values at

1 nm intervals. This was essential, as 1 nm steps were needed for input into the

Dichroweb internet based CD data analysis program. The spectrum was collected 5

times over this range so reproducibility could be evaluated.

Protein thermal denaturation protocol

The samples were prepared and spectra collected as previously stated. The temper-

ature range examined was from 5℃ to 70℃ and as it was lowered back to 10℃ in

5℃ increments. The temperature was allowed to stabilize and sample to adjust to

each temperature for 30 seconds, data acquisition completed for each temperature and

the temperature adjusted again. Data collection time was minimised to decrease the

effects of extended incubation at an elevated temperature.

Data analysis

Dichroweb (www.cryst.bbk.ac.uk/cdweb/html/home.html) - a subscription-based cir-

cular dichroism data analysis website - was used for data analysis. Data inputs required

were wavelength and millidegrees of ellipticity. The CDSSTR algorithm and reference

set 7 which was the most suitable for the data values were selected. CDSSTR im-

plements the variable selection method by performing all possible calculations using

a fixed number of proteins from the reference set. The algorithm recognises proteins

possessing characteristics not reflected by the test protein or proteins not reflecting the

47

characteristics of the test protein, and removes them from the basis set. It then uses

the SVD algorithm which assigns secondary structure. This method was recommended

to be paired with the Reference Set 7 of protein samples.

2.11.3 Crystallisation trials

Protein, including that used for analysis by CD and DLS, was concentrated to 9 mg.mL−1

and any precipitant cleared by a 5 minute spin on a desktop microcentrifuge at approx-

imately 14,000g and 4℃. This protein was kept on ice, and hanging drops set up on

a Mosquito crystallization robot using a 96 well plate format. The initial screen used

was The Joint Consortium for Structural Genomics Screen as provided by Hampton

research. Hanging drops with a total volume of 200 nL were used, comprising of 100

nL of the well solution and 100 nL of the protein sample. Drops were examined under

the microscope to determine an initial condition for each sample. This was done in

duplicate for each condition; one tray was stored at 4℃ and the other at 18℃. The

trays were then examined for crystals on the second day after their creation, then

weekly thereafter. Further screens were set up as outlined in the results chapter on

crystallisation trials (§ 5.3) and the discussion (§ 6.8.3).

48

2.12 Computer Software

Table 2.13 shows the computer software packages used for the analysis of data gathered

as disclosed herein and subsequent production of this manuscript.

Table 2.13: Computer Software

Title Description

Enzyme X 3.0 A program made by Alexander Griekspoor and Tom Groothuis. De-

signed to help find the perfect restriction enzymes for desired cleavage

of DNA.

DNA Strider1.4f6 DNA and protein manipulation software.

Adobe Photoshop CS 8.0 Photoediting software used for correcting contrast and balance of gel

photos and cropping to correct size.

OmniGraffle Used for creating all figures in this manuscript.

MacPyMOL 0.99 Protein modelling software used to determine locations of domains

of TF and to help choose locations for truncations.

4Peaks 1.7.1 Software for analysis of DNA sequencing files.

Sequencher 4.5 Demo Software for analysis of DNA sequencing files and the allignment of

the files.

TeXShop 2.26 Publishing and typesetting software produced under GPL public li-

cense.

Microsoft Excel 11.3.6 Spreadsheet and graphing software produced by Microsoft Corpora-

tion, 2004.

49

Chapter 3

Creation and physiological analysis

of Synechocystis sp. PCC 6803

TFSpec

The primary goal of this set of experiments was to create a Synechocystis sp. PCC 6803

TFSpec mutant. This strain would be used to examine the phenotype of the mutant

and compare it to the cold sensitivity phenotype seen in E. coli (Kandror and Goldberg

(1997)). It would also be used to examine the rate of Photosystem II recovery after

photoinhibition.

The strategy was to amplify the TF gene and insert the spectinomycin resistance

gene into the coding region, creating an interruption. Transformation of this construct

into Synechocystis allows the replacement of the genomic copy of TF by homologous

recombination and subsequently, selection of the knockout.

3.1 Creation of Synechocystis sp. PCC 6803 TFSpec

3.1.1 Sequence acquisition and primer design

The sequence for the TF gene was obtained from the CyanoBase website

(http://www.kazusa.or.jp/cyano/) and a search in the Synechocystis database done.

The sequences in the database are annotated for predicted genes and known genes, the

51

search by name yielded one copy of the Trigger Factor (TF) gene in the genome. This

was essential as other functionally similar genes in Synechocystis have multiple copies,

for example, there are three copies of the chaperone DnaK (Varvasovszki et al. (2003)).

The gene TF is found at locus number sll0533 and primers designed as described in

Eaton-Rye (2004).

Primers were chosen so that approximately 1 kb either side of the antibiotic resis-

tance cassette was to be amplified, and examined in the program Amplify 3X to check

for unsuitable characteristics such as the potential to form primer dimers.

Figure 3.1: ORF map of Synechocystis sp. PCC 6803 in the region around TF (locus sll0533). Shown

along the top is the position of the gene in the genome, below are the ORFs in the region of TF and

their direction as indicated by the arrowhead.

PCR cloning of TF

Genomic DNA was extracted from Synechocystis using the method described in § 2.5.3.

DNA extraction from a 3.4 g pellet of cells yielded 4.1 µgµL−1 DNA. PCR was done

using primers TF forward and TF reverse (see Table 2.11 for sequences). Figure 3.2

shows the PCR product that was obtained using the PCR protocol in Table 2.8 and

the manufacturer’s instructions. The desired product can be seen at 2 kb.

The PCR product was purified using the PCR purification method described in

§ 2.5.6, this yielded the DNA with the concentration of 45 ngµL−1 and a A260/A280 of

1.81.

52

1

3

1.62

Size

in k

B

Mar

ker

TFFigure 3.2: PCR cloning of TF from genomic DNA. A 4 µL portion of the PCR reaction (Lane TF)

was run on a 0.8% agarose gel as described in § 2.6.1. The Marker lane contains 5µL of 1kb DNA

ladder (prepared as outlined in § 2.6.1). Marker sizes are shown on the left.

The purified PCR product was ligated into the plasmid pGEM-T using the Promega

pGEM-T™Vector System according to the manufacturer’s instructions using a 3:1 ratio

of insert to vector.as described in § 2.5.9. This is possible as Taq DNA polymerase

creates a ‘T’ overhanging base that aids the insertion of the product into the plasmid

which has a ‘A’ overhang.

Plasmids were prepared (§2.5.1) from selected transformants (§2.3.6) and analysed

by restriction digest with XmaI and run on a 0.8% agarose gel. This gave the expected

restriction digest pattern of a single band of about 5.1kB.

3.2 Insertion of Spectinomycin resistance into TF

Spectinomycin cassette purification

The spectinomycin cassette was obtained from the XmaI digest of lab stock pUC19

plasmid, grown (§ 2.3.1) and purified (§ 2.5.1) as described. Separation on a 0.8%

agarose gel and extraction (§ 2.5.2) yielded 870 ng with an A260/A280 of 1.82.

53

Ligation of Spectinomycin into TF and transformation into DH5α cells

The purified DNA fragment containing the spectinomycin resistance cassette was lig-

ated into the pGEM-T TF plasmid cut with XmaI and dephosphorylated with SAP

as described in the manufacturer’s instructions. This was done with a ratio of 3:1,

Insert:Vector (§ 2.5.9). Transformants in DH5α cells (§ 2.3.6), and these were selected

on LB-spectinomycin/ampicillin plates overnight as per § 2.3.1.

Restriction digest and analysis

Plasmids purified from two elected transformants were then digested with SphI for

analysis. The pattern expected to be seen for the pGEM-T TF was a single band at

5 kb, the pattern expected to be seen for the digest or pGEM-T TFSpec depended on

the orientation of both the TF and the spectinomycin inserts. The combinations were

determined that the product sizes seen, 4 kb and 3 kb correlated with both TF and

Spec inserts in the reverse direction. Figure 3.3 shows the plasmid map of pGEM-t

TF containing the Spectinomycin resistance cassette and Figure 3.4 shows the agarose

gels of the experimental digest.

Sequencing of pGEM-t TFSpec resistance plasmid

To confirm the identity of the construct the plasmid was sequenced, primers compli-

mentary to promoter regions SP6 and T7 (§ 2.6.3). The sequence and chromatograms

confirmed the presence of the TF gene and the interruption of the ORF with the

spectinomycin resistance cassette (See appendix).

3.2.1 Transformation of cyanobacteria

The plasmid was then transformed into WT Synechocystis (§ 2.3.7). Transformants

were grown under the selection of spectinomycin and restreaked weekly, with segrega-

tion monitored by colony PCR (§ 2.5.5) using a template from a very small amount

of cells (just enough to cover the end of a 20-200 µL disposable pipette tip. When it

appeared, after about 6 weeks, that there were no WT copies of the genome present

(Figure 3.5), a southern blot was done (Figure 3.6).

54

Figure 3.3: Plasmid map of pGEM-T TFSpec. The TF gene, flanking regions and the spectinomycin

resistance cassette are indicated

55

1

3

1.62

45

Size

in k

B

Mar

ker

Cut

Uncu

t

1

3

1.62

45

Size

in k

B

Mar

ker

Cut

Uncu

tFigure 3.4: pGEM-t TF vector before and pGEM-t TFSpec after the insertion of the spectinomycin

resistance cassette. On the left: TF in the pGEM-t vector cut with SphI. Marker: 1kb DNA ladder,

Uncut: uncut plasmid, and Cut: plasmid cut at a single SphI restriction site using the method as

described in§2.5.8. On the right: The restriction digest of the pGEM-t TFSpec plasmid after the

insertion of the spectinomycin resistance cassette. Marker: 1 kb DNA ladder, Cut: plasmid cut with

SphI as described in §2.5.8, Uncut: uncut plasmid. On the left hand side of both figures are the size

of the molecular markers.

56

Colony PCR

Colony PCR shows the TFSpec interruption PCR product running at a size of just

less than 4 kb compared to WT (1.6kb), consistent with the insertion of the 2 kb

spectinomycin resistance cassette (Figure 3.5). Figure 3.5 illustrates the result of the

PCR showing that there are no WT copies of the genome present.

Southern blot

The genomic DNA (5mg) was digested overnight with PstI and EcoRI in Buffer H

(§ 2.5.8), separated on a 0.8% agarose gel (§ 2.6.1) and transferred to the nitrocellulose

membrane (§ 2.7.1). A probe was created (§ 2.7.2) using SacI and BglII to create the

appropriate template from the pGEM-t TFSpec plasmid. The expected size of WT (1.6

kb) and the TFSpec (3.6 kb) were observed (Figure 3.6). It is also noted that there

is no band, relating to the WT sequence, at 1.6 kb in the lane of the TFSpec mutant.

Figure 3.6 confirms the colony PCR result, showing that there are no WT copies of

the genome present.

57

1

3

1.62

4

Size

in k

B

Mar

ker

WT

TF-S

pec

Figure 3.5: Colony PCR of the TFSpec interruption and WT. Marker: 5 µL of 1kb DNA ladder,

TFSpecSpec: 4µL of the TFSpec interruption PCR product, WT: 4µL of the WT PCR reaction.

Samples are run on a 0.8% Agarose gel as described in § 2.6.1 and stained with a standard concentration

of EtBr.

58

1.6

3

8

Mar

ker

WT

TF-S

pec

Size

in k

B

Figure 3.6: Southern blot of the TFSpec interruption and WT. Marker: 1kb DNA ladder, WT: WT

genomic DNA digest with PstI and EcoRI, TF-Spec: TFSpec interruption genomic DNA digest with

PstI and EcoRI. This was done in line with the method outlined in § 2.7. Film was exposed for 48 h

at -80℃.

59

3.3 Physiological studies

Physiological studies were carried out comparing the TF knockout mutant of Syne-

chocystis to the well characterised WT.

3.3.1 Growth rate

Synechocystis cells were grown on plates as described in § 2.3.1 and individual cell

samples were spread onto three individual plates and grown for several days. A liquid

cultures derived from each individual plate was then prepared as described in § 2.4.1.

Figure 3.7 shows the growth rate of both strains of cells at 18℃ and 30℃. The growth

rates of the TFSpec strain is lower than the WT strain in both of the temperature

conditions. Each O.D. was measured below a value of 0.4 by diluting the sample, the

measured O.D. was then multiplied by the dilution factor.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 13 26 36 48 60 72 84 96 108 120 132 144 156 168

Time (hours)

Op

tica

l D

en

sity

(7

30

nm

)

TF 18 WT 18

TF 30 WT 30

Figure 3.7: Growth curves of TFSpec and WT at 30℃ and 18℃. Error bars represent the standard

error of the mean of triplicate measurements.

60

3.3.2 O2 evolution

The photosynthetic capacity of the TFSpec strain was assessed by its ability to evolve

O2 at moderate light intensities. Cells were adjusted to 10 g.mL−1 and the O2 evolution

was measured using the Oxygen electrode and method as described in § 2.4.3 and

§ 2.4.4. Measurements were done in triplicate on three independently grown cultures

for both WT and the TFSpec strain. Results were then converted to a percentage of

the initial rate to allow comparisons. The TFSpec strain showed two differences from

WT. The slope, shown in Figure 3.8, of O2 evolution by the TFSpec strain is slower

that for he WT strain over the same period. Also worthy of note is a marked inflection

point of the TFSpec strain showing a decrease in the rate at about 30 s after the start

of illumination.

0.98

1

1.02

1.04

1.06

1.08

1.1

1.12

1.14

1.16

0 60 120 180 240

Time (s)

Perc

en

tag

e c

han

ge o

f O

2 e

vo

luio

n

WT strainTF- strain

Figure 3.8: Oxygen evolution of WT and TFSpec Synechocystis cells. The vertical line at 60 s indicates

the time point that the light was turned on and the cells start evolving O2.

61

3.3.3 Photoinhibition

The ability of the TFSpec strain to recover from photoinhibition was evaluated by its

ability to evolve O2 after exposure to high light intensities this is shown in Figure 3.9.

Cells were grown and treated as described in § 2.4.2 and samples were analyzed in the

Oxygen electrode as described in § 2.4.4. The raw experimental data was converted to a

percentage of the initial rate of evolution by division of each experimentally determined

value by the initial value to allow a comparison over time of the samples and for the

calculation of meaningful statistics (Standard error of the mean) and t-test. Both

strains showed an initial decrease in the O2 evolution rate with a slight recovery by

the 45 min mark. The TFSpec recovered statistically significantly more slowly than the

WT (p values of the difference between TFSpec and the WT strains being 0.0088 at 75

min and 0.025 at 90 min) but may eventually recover to a higher level, although this

is later difference was not found to be statistically significant.

62

0

20

40

60

80

100

120

140

160

180

0 45 90 135

Time (Minutes)

Perc

en

tag

e o

f t=

0 (

%)

TF Average %

WT Average %

Figure 3.9: Photoinhibition of TFSpec and WT Synechocystis cells. The vertical line at 45 min

indicates the time point that the light was turned off and the cells returned to 30 µE m−2s−1. Error

bars represent the standard error of the mean of triplicate measurements.

63

3.4 Truncations of TF

This section describes the initial step of the construction of genes for truncated versions

of TF. The discernible physiological differences between TFSpec and WT Synechocystis

cells make it feasible to test the contribution of each domain of the protein. More

specifically, we intend to determine whether deletion of certain domains of the protein

showed the same phenotype as WT or TFSpec strains.

Strategy

The crystal structure of Vibro cholerae TF was retrieved from the PDB (accession

1T11) and domain boundaries determined as outlined in Ludlam et al. (2004). The

domain boundaries of Synechocystis TF were determined by using clustalw to create a

multiple alignment of the Synechocystis TF protein sequence with the Vibro cholerae

TF protein sequence. This allowed the determination of the nucleic acid sequences that

coded for the domain boundaries and the design of primers to amplify the domains and

flanking regions as demonstrated in Figure 3.10. Initially a TF deletion mutant needed

to be made to ensure complete replacement of the TF gene with the mutated version.

Once this was made, and the WT copy of the genome eliminated from the Synechocys-

tis cells then transformation with the truncated versions of TF could be done.

Domain 1 (residues 1-113) is at the N-terminal of the protein directly connected to

domain 3 (residues 114-150 and 248-379) which is a spatially defined domain and is

broken by domain 2 (residues 151-247) (Figure 3.11). To design a way for the trunca-

tions to be made in any combination, palindromic hexanucleotide restriction enzyme

sequences with identical four central nucleotides were identified. These were designed

into the central region of PCR primers spanning each of the domains boundaries (Fig-

ure 3.10).

Primers were designed to include flanking regions of the gene to allow homologous

recombination into Synechocystis. The enzymes that suited this with an identical four

nucleotide central region were AvrII, SpeI, NheI and XhoI. AvrII could not be used

due to there being an AvrII site in the plasmid that was required. Further to these

64

restriction endonucleases, two blunt-end generating restriction sites were used, EcoRV

and StuI, these were selected due to their absence in the plasmid and the gene and an

increased existing complementarity to the existing genomic sequence.

3a 1 2 3b

A B G

h g e d f c

Figure 3.10: TF truncation primer positions. Primer positions and directions are indicated. Details

of the particular features of the primers are shown in Table 2.11. 1 refers to Domain 1, 2 refers to

Domain 2, 3a and 3b refer to the two parts of Domain 3.

3.4.1 PCR of TF domains

Insertion into pGEM-t amplification A-h

Initial attempts at amplification of the individual domains of TF from genomic DNA

yielded multiple unwanted products in the process of the PCR. Hence, the whole TF

gene and the surrounding region was amplified using specifically designed primers and

inserted into a pGEM-5, this was then used as the template for the further amplifi-

cations of the different domains of TF. The primers had to be designed to remove as

much of the restriction cassette of the vector as possible as the pGEM-5 restriction

cassette contained several of the desired restriction sites for the final construct. These

restriction sites inserted in the gene needed to be unique so they could be selectively

excised and ligated to construct the various truncated versions of TF. The PCR prod-

uct using primers for the entire flanking region (primer A and primer h (Table 2.11))

were used to generate the insert. This was then ligated into a SphI and SalI) digested,

65

Figure 3.11: TF domain boundaries. TF protein sequences from Vibro cholerae and Synechocystis

were aligned using the clustalw multiple alignment algorithm and the domain boundaries were overlaid

from the Vibro cholerae. Shown in blue is domain 1 with the gold de-marking the ribosome binding

region, green is the domain 2, the chaperone domain and magenta is domain 3, the PPIase domain.

66

purified pGEM-5 vector using the ProMega pGEM-t ligation kit.

Primer A-c amplification

The 5’ flanking region of the gene region was first amplified using the appropriate

primers (Primer A and Primer c (Table 2.11)) and using the previously made pGEM-5

A-h as the template. This yielded the appropriate product that was then isolated as

described in § 2.5.6.

3.4.2 Mega-primer PCR

Primer Ac-g

Initially, the desired method for the creation of the plasmid for the deletion of TF was

to introduce restriction digest sites at the 5’ and 3’ ends of the TF gene. From this,

the gene its self could be then deleted and a selection cassette could be inserted. This

would also provide the most logical starting point for the introduction of the restriction

sites at the domain boundaries.

The product from the amplification of primers A-c was then used as the primer for the

amplification of the fragment termed Ac-g. This was done using pGEM-5 A-h as the

template, PCR product A-c as the forward primer and primer g as the reverse primer.

The annealing time and temperature was increased to increase the binding potential

of the longer primer. Extension time and temperature remained normal. This yielded

the correct product of 1.4 kb can be seen in Figure 3.12. It also shows a strong PCR

product of unknown identity at about 850 kb and the A-c primer at 0.4 kb.

Primer Acg-h

It was next attempted to amplify the section Acg-h, using Ac-g as the forward primer,

purified via gel extraction from the previous round of amplification, and h as the reverse

primer. The product of this final amplification would provide the basis for deletion of

the TF ORF by the insertion of the Chloramphenicol resistance cassette into the place

of the gene. The amplification of this product was attempted under an extensive range

67

Mar

ker

PCR

prod

ucts

21.6

1Si

ze in

kB

0.5

3

Figure 3.12: PCR product of the amplification of Ac-g seen at 1.4 kb and the remnants of the A-c

primer at 0.4 kb

of conditions but the difficulty of obtaining starting primer and the evasiveness of the

correct conditions for the amplification led to this approach being abandoned.

Primer G-h amplification

This was used to create the downstream flanking region of TF for ligation of the

Chloramphenicol resistance into a pGEM-t construct. A new primer was designed to

be complimentary to the primer g. This ensures that the restriction sites that were

designed into the system remained as desired. The amplification of the G-h product

was done using a standard PCR protocol and purified using standard techniques.

Chloramphenicol resistance cassette amplification

The chloramphenicol resistance cassette was amplified using primers designed to have

the appropriate restriction digest sites, these were known as Chl fwd and Chl rev. They

have a short extension at the end of the primer that serves to aid the annealing to the

template and to allow ample room for the restriction enzymes to cleave the recognition

sequence.

68

3.4.3 Ligation and analysis

All four segments, the digested pGEM-5 vector, the A-c fragment, the chloramphenicol

fragment and the G-h fragment, were ligated in a single reaction in approximately

equimolar amounts. After selection and analysis by restriction digest this gave a digest

pattern with SalI and SphI that had bands at approximately 3 kb and 1.4 kb. It was

found, when compared to the theoretical construct size that this product was about 450

bp short indicating that the downstream flanking region was missing from the product.

This was able to be cut with the appropriate enzymes and attempted to insert the A-c

product. All attempts to insert this fragment failed.

Sequencing

Upon sequencing with the T7 and SP6 primers it was seen that the required restriction

site was present in the construct but was predicted with a very low certainty. Using

these primers would have amplified both of the flanking regions strongly, however, on

the 5’ end of the forward direction sequencing product showed the Chloramphenicol

resistance cassette. This was interpreted to confirm the absence of the G-h flanking

region. See the chromatograms in the appendix for the sequences.

69

Chapter 4

Antibody creation and testing

4.1 Overview

To detect whether Trigger Factor (TF) is involved in the repair of D1, antibody pull-

down experiments were chosen to detect the presence of TF bound to thylakoid mem-

brane associated ribosomes. The prior art indicates (de Keyzer et al. (2003); Raine

et al. (2004); High et al. (1997)) that the SRP may have an involvement in the repair

of D1 and potentially also associates with TF.

Further to investigating the involvement of TF with D1, and given previous studies

finding a potential association between TF and the SRP, the D1 protein in Synechocys-

tis sp. PCC. 6803 was considered to be a good model to find such interaction between

TF and the SRP due to the high abundance of D1 and the requirement for the cells to

replace this protein quickly and efficiently. In Synechocystis sp. PCC. 6803 the protein

FFH (Fifty-Four Homologue) is the fifty four kDa subunit homologue of the eukaryotic

SRP (Gutierrez et al. (1999)).

Antibodies to the TF protein from Mycobacterium tuberculosis, Escherichia coli,

Heamophilus influenzae and Aquifex aeolicus were available and were tested against a

cell lysis of Synechocystis sp. PCC. 6803. No reactivity was detected to a polypeptide of

the correct molecular weight for TF. No antibody was available for either Synechocystis

sp. PCC. 6803 TF or FFH.

To create antibodies to TF and FFH from Synechocystis sp. PCC. 6803 the coding

71

region for each gene was amplified and inserted into the pENTR-11 GateWay Entry

vector. This can then be recombined with the GateWay pDEST vectors to allow ex-

pression and purification of the protein fused with an affinity tag and a 3C protease

sequence to remove the tag after purification. Different expression cell lines were as-

sessed for the greatest amount of soluble protein produced. Purified, concentrated

protein was used for antibody production by subcutaneous injection into rabbits for

the production of rabbit Anti-TF and rabbit Anti-FFH antibodies.

To investigate the role of TF in the replacement of D1 in Synechocystis sp. PCC.

6803 pull down experiments were attempted to show an interaction between TF, FFH

and D1. This was done on high light, normal light and dark prepared cells.

4.2 Cross reactivity of TF antibodies

4.2.1 Analysis of cross reactivity of the Trigger Factor anti-

bodies

Detection of Trigger factor associated with ribosomes in Synechocystis sp. PCC. 6803

is a central part of the investigation into finding association with TF and the nascent

D1 protein. The Wilbanks lab already had antibodies raised against A. aeolicus, M.

tuberculosis, E. coli and H. influenzae trigger factor (sources are listed in Table 2.12). If

the interaction of one or more of these antibodies could be established an investigation

into the interaction of TF with D1 could commence. The TF antibodies are polyclonal

and due to this, some cross-reactivity with highly abundant proteins could be expected.

4.2.2 Phylogenetic analysis of Trigger Factor proteins

To assess the relatedness of the different trigger factor proteins they were aligned by

the alignment program clustalw (Thompson et al. (1994)) and a phylogenetic tree was

created with kitsch (Felsenstein (1993)). The kitsch algorithm estimates phylogenies

from alignment data where the distances are expected to equal the sums of branch

lengths between the species except that an evolutionary clock is assumed. This showed

72

that the Synechocystis sequence is not obviously more closely related to one family of

the bacteria. Hence, it is not predictable which, if any, of the antibodies was likely to

be the most cross-reactive.

Figure 4.1: A kitsch tree of the Trigger Factor amino acid sequences. The alignment was done using

the Amino Acid Maximum Likelihood program using the Jones-Taylor-Thornton model of amino acid

change. Accession numbers: H. influenzae AAC22370, E. coli NP 752485, M. tuberculosis CAA16039,

Synechocystis sp. PCC 6803 NP442797 and A. aeolicus NP213922.

4.2.3 Analysis of TF antibodies from A. aeolicus, M. tuberculosis,

E. coli and H. influenzae

Western Blots were done to ascertain whether antibodies raised against TF proteins

from other species were crossreactive with Synechocystis sp. PCC. 6803 TF (§2.9.2

and §2.9.3). The primary antibodies used were Rabbit anti-TF from Aquifex aeolicus,

Escherichia coli, Heamophilus influenzae and Mycobacterium tuberculosis. Binding of

the antibodies was detected by a chemo-luminescent reaction via a secondary goat-

anti rabbit antibody with a conjugated Horse-radish Peroxidase enzyme. Western blot

analysis is shown (Figure 4.2) showed no cross reactivity to protein of the predicted

size for TF. The 10 mg.mL−1 lane analysed with the A. aeolicus antibody reacts to a

large clump of proteins at about 200kD with a ladder of other proteins down to about

90kD. No reactivity can be seen for either concentration of E. coli or H. influenzae

antibodies, M. tuberculosis demonstrates weak reactivity at approximately 21kDa.

73

41

80

201

Mar

ker

10m

g.m

L-1

1mg.

mL

-1

Size

in k

Da

128

31

a.

41

80

201

Mar

ker

10m

g.m

L-1

1mg.

mL

-1

Size

in k

Da

128

31

b.

41

80

201

Mar

ker

10m

g.m

L-1

1mg.

mL

-1

Size

in k

Da

128

31

c.

41

80

201

Mar

ker

10m

g.m

L-1

1mg.

mL

-1

Size

in k

Da

128

31

d.

Figure 4.2: Western analysis of Synechocystis sp. PCC. 6803 Trigger Factor with antibodies raised

against 4 different species TF proteins. The primary antibodies used were Rabbit anti-TF from

Aquifex aeolicus (Figure 4.2a), Escherichia coli (Figure 4.2b), Heamophilus influenzae (Figure 4.2c)

and Mycobacterium tuberculosis (Figure 4.2d). Two different concentrations of cellular extract were

used. In each panel, lanes were loaded as follows: Marker: Kaleidoscope molecular weight maker,

10 mg.mL−1: 10 mg.mL−1 of lysed Synechocystis sp. PCC. 6803 , 1 mg.mL−1: 1 mg.mL−1 of lysed

Synechocystis sp. PCC. 6803. Antibody concentration used as indicated in Table 2.12

74

4.3 Protein expression and purification using the

Gateway system

4.3.1 Amplification of TF and FFH for pENTR 11

To generate the pENTR-11 TF and the pENTR-11 FFH vectors , primers were designed

to amplify the coding region of each gene. In both cases a 3C protease site (CTG

GAA GTT CTG TTC CAG GGG CCC) was included at the 5’ end of the forward

primer. For insertion into the pENTR-11 vector, a XhoI restriction endonuclease site

was added immediately after the stop codon along with 4 nucleotides corresponding to

the flanking region of the gene. A large scale genomic DNA preparation was done as

described in §2.5.3 and used as the template for the amplification of TF and FFH and

shown in Figure 4.3 and purified (§2.5.6). PCR of TF (sll0553) was done using primers

TF GateWay forward and TF GateWay reverse (Table 2.11) using genomic DNA as

a template and the standard PCR protocol outlined in §2.5.5 but with an annealing

temperature of 60℃. FFH (slr1531) was amplified using primers FFH GateWay forward

and FFH GateWay reverse (Table 2.11) in the same conditions as TF stated above.

The PCR products were purified as described in §2.5.6.

Mar

ker

TF 6

0°C

TF 6

5°C

FFH

60°C

FFH

65°C

1.62

1

0.5

34

Size

in k

B

Figure 4.3: PCR products of TF and FFH genes. PCR primers pair and anneal, temperatures are as

indicated.

75

The pENTR-11 vector was prepared in E. coli DB3.1 cells by others and the plasmid

isolated as described in §2.5.1. This plasmid was sequentially digested to remove the

DH5α lethal ccdB gene. The initial digest was done with XmnI in NEB Buffer 2 then

heat killed at 95℃. The salt concentration was adjusted with Buffer M to H conversion

buffer (1µL per 10 µL reaction, Table 2.5.8) and 0.5µL SuRE Buffer H then the total

volume made to 15µL with MQ H2O and 0.5µL XhoI and digested overnight. The TF

and FFH inserts were then ligated with this vector and plasmids from transformants

were screened with a PstI digest (Figure 4.4). A single band was expected at 3.8kb for

both of the PCR products, these can be seen in the TF digest and FFH digest lanes

for one of two clones for each construct.

Mar

ker

TF cl

one d

iges

t

FFH

clone

dig

est

1.61

2

0.5

34

Size

in k

B

TF cl

one d

iges

tFF

H clo

ne d

iges

t

Figure 4.4: PstI digest of pENTR-11 vector with TF and FFH insert. The digest was done in the

usual manner as described in §2.5.8. Marker: 1kb DNA ladder, TF clone digest: TF clone digested

with PstI, TF clone digest: TF clone digested with PstI showing unknown digest pattern, FFH clone

digest: FFH clone digested with PstI showing unknown digest pattern, FFH clone digest: FFH clone

digested with PstI

4.3.2 Recombination into pDEST vectors

Plasmids that gave the desired PstI digest were selected and recombined with the

pDEST-15, pDEST-17 and pET(gwA)-32 vectors as described in §2.6.4. These were

selected via ampicillin resistance, amplified as per §2.5.1 and screened with a XmnI

restriction digest (Figure 4.5) This indicated that eight out of the ten selected colonies

76

displayed the predicted restriction digest pattern. The clones labelled pET(gwA)-32

FFH clone 1 and pET(gwA)-32 FT clone 1 did not display the predicted restriction

digest pattern.

Mar

ker

pDES

T-15

TF

clone

1

pDES

T-15

FFH

clo

ne 1

pET(

gwA)

-32 T

F clo

ne 2

pDES

T-17

TF

clon

e 1

pDES

T-15

FFH

clon

e 2pE

T(gw

A)-3

2 FF

H clo

ne 2

pDES

T-15

TF

clone

2pD

EST-

17 T

F clo

ne 2

21.6

1

3

Size

in k

b

pET(

gwA)

-32

FFH

clon

e 1pE

T(gw

A)-3

2 TF

clon

e 1

Figure 4.5: Screening of the recombination products of pENTR-11 FFH and pENTR-11 TF vectors

into the pDEST-15, pDEST-17 and pET(gwA)-32 vectors. All samples were digested with XmnI and

run on an 0.8% agarose gel. Marker: 1kb DNA ladder, remainder of the lanes are labelled as to the

plasmid digest portrayed.

4.4 Trial expression

Trial expression was done to determine the cell line that had the greatest expression of

soluble recombinant protein. This was done in E. coli BL21 (DE3) and E. coli BL21

(DE3) Star cells (see §2.6 and §2.3.6 for details) that were transformed with pDEST-15

(GST tag), pDEST-17 (His tag) and pET(gwA)-32 (Trx-His tag) as created in §4.3.2.

The cells were grown at 37℃, induced (§2.8.1) and the cells lysed (§2.8.2). Figures 4.6 to

4.10 below show the expression trials of TF in pDEST-15, pDEST-17 and pET(gwA)-32

and FFH in pDEST-15 and pET(gwA-32).

Figure 4.6 shows that there is expression of protein for both cells types trialed,

however the majority of the protein expressed in the BL21(DE3) cells is insoluble and

that, although more soluble in the BL21(DE3) Star cells there is only a small amount

of soluble protein present.

77

Figure 4.6: Trial Expression of Trx-His-TF in BL21 (DE3) or BL21 Star cells. The cells were trans-

formed, grown, induced and lysed as described in §4.4. Samples, labelled along the top as Uninduced,

Uninduced and Soluble, were prepared as described in §2.8.2 for TF expressed from either pET(gwA)-

32 in BL21 (DE3) cells or BL21 Star cells as indicated under the lanes. The lane labelled Marker

contains Low-Mass SDS-PAGE marker. The expected mass of the recombinant TF-tagged protein is

indicated with the arrow.

78

Figure 4.7: Trial Expression of GST-TF in BL21(DE3) and BL21Star cells. The cells were trans-

formed, grown, induced and lysed as described in §4.4. Samples, labelled along the top as Uninduced,

Uninduced and Soluble, were prepared as described in §2.8.2 for TF expressed from either pDEST-15

in BL21(DE3) cells or BL21 Star cells as indicated under the lanes. The lane labelled Marker contains

Low-Mass SDS-PAGE marker. The expected mass of the recombinant TF-tagged protein is indicated

with the arrow.

79

Figure 4.7 shows that there is expression of protein for both cells types trialed, the

majority of the protein expressed in both cell types is soluble however, the GST-TF

protein expressed in BL21(DE3) cells is clearly more abundant.

Figure 4.8: Trial Expression of His-TF in BL21 (DE3) and BL21 Star cell. The cells were transformed,

grown, induced and lysed as described in §4.4. Samples, labelled along the top as Uninduced, Unin-

duced and Soluble, were prepared as described in §2.8.2 for TF expressed from either pDEST-17 in

BL21 (DE3) cells or BL21 Star cells as indicated under the lanes. The lane labelled Marker contains

Low-Mass SDS-PAGE marker. The expected mass of the recombinant TF-tagged protein is indicated

with the arrow.

Figure 4.8 shows that there is poor expression of protein for both cells types trialed,

there is observable protein expressed in the BL21(DE3) Star cells however there is only

a small amount of soluble protein present.

Figure 4.9 shows that there is a large amount of expression of protein for both cells

types trialed and the majority of the protein expressed in both cell types was soluble.

Figure 4.10 shows that there is poor expression of protein for both cells types trialed,

there is observable protein expressed in both cell types however there is only a small

amount of soluble protein present.

To choose the optimal expression vector for production of the respective proteins in

80

Figure 4.9: Trial Expression of Trx-His-FFH in BL21 (DE3) and BL21 Star cell. The cells were

transformed, grown, induced and lysed as described in §4.4. Samples, labelled along the top as

Uninduced, Uninduced and Soluble, were prepared as described in §2.8.2 for FFH expressed from

either pET(gwA)-32 in BL21 (DE3) cells or BL21 Star cells as indicated under the lanes. The lane

labelled Marker contains Low-Mass SDS-PAGE marker. The expected mass of the recombinant TF-

tagged protein is indicated with the arrow.

81

Figure 4.10: Trial Expression of GST-FFH in BL21 (DE3) and BL21 Star cell. The cells were

transformed, grown, induced and lysed as described in §4.4. Samples, labelled along the top as

Uninduced, Uninduced and Soluble, were prepared as described in §2.8.2 for TF expressed from either

pDEST-15 in BL21 (DE3) cells or BL21 Star cells as indicated under the lanes. The lane labelled

Marker contains Low-Mass SDS-PAGE marker. The expected mass of the recombinant TF-tagged

protein is indicated with the arrow.

82

a larger scale, both yield and ratio of soluble to insoluble protein were considered. The

expression construct chosen to express TF is the pDEST-15 vector in BL21 (DE3) cells

(Figure 4.7) expressing an N-terminal GST-fusion to TF. This gave the highest expres-

sion and proportion of soluble protein to insoluble protein. The expression construct

for FFH was chosen for the same characteristics. Therefore the pET(gwA)-32 vector

in BL21(DE3) cells was chosen (Figure 4.9) expressing N-terminal Trx-His fusion to

FFH.

4.5 Large scale purification of TF with GST tag

(pDEST-15)

TF with an N-terminal GST tag was expressed in BL21(DE3) cells as described in

§2.8.1, prepared (§2.8.2) and applied to GST resin (§2.8.3). Induction of TF produced

a large amount of soluble protein (see Clarified lysate lane, Figure 4.11). A large

proportion of protein did not bind to the resin (Unbound fraction).

Soluble protein was removed after 24 hours 3C cleavage of TF from the GST resin

in a total volume of 5 mL. The cleaved fraction was separated over by Size Exclu-

sion Chromatography (§2.8.5) over a Superdex 200 column. Samples of the fractions

showing the highest A260 trace were then run on a 12% acrylamide gel as described

in §2.9.1 (Figure 4.11). The TF eluted over a broad range from column fractions 9-13

correspond to a total volume of 20mL.

Fractions from SEC lanes 10 to 12 shown in Figure 4.11 were concentrated using

the method outlined in §2.8.6 from a total volume of 12 mL to a final volume of 1.2

mL with a concentration of 1.4 mg.mL−1 (Figure 4.12 B, Concentrated fraction). No

protein was detected in the flow through fractions (data not shown).

There was a large amount of full length TF-GST recombinant protein still bound

to the resin after 24 hours of cleavage. Therefore, the resin was incubated for a further

48 hours and the washed 3 times with PBS and three times with PBS and 10 mM

glutathione (Figure 4.12 A). Cleaved product was pooled (Figure 4.12 B, 72 Hour

3C digest) and compared by SDS-PAGE with the concentrated protein purified by

83

9666

44

32

22

14

Size

in k

Da

Mar

ker

Unin

duce

dIn

duce

dLy

sed

Cells

Clar

ified

Lys

ateGS

T re

sin af

ter 3

C cle

avag

e

Unbo

und

bef

ore c

leava

ge

Supe

rnata

nt af

ter cl

eava

ge

9 10 11 12 13

Superdex 200 Column Fractions

Figure 4.11: Analysis of the expression and purification of TF. Uninduced, induced and lysate indicates

whole cells treated as described (§2.8.1 and §2.8.2). Clarified lysate and Unbound before cleavage

indicate protein solution before and after binding to the GST resin. GST resin after 3C cleavage

shows all products of cleavage, while Supernatant after cleavage shows only soluble products. Last

five lanes show selected fractions from gel filtration column. Marker sizes (Low-Mass SDS-PAGE

marker) are as indicated on the left.

84

Superdex. The samples taken at 72 hours were seen to be as pure and at a higher

concentration than the protein that was cleaved and purified by SEC (Figure 4.12 B).

Mar

ker

96

66

42

3222Si

ze in

kD

a

14

Was

h 1

Was

h 2

Was

h 3

Elut

ion

1El

utio

n 2

Elut

ion

3Re

sin sa

mpl

e

72 h

our 3

C di

gest

Conc

entra

ted fr

actio

n

A B

Figure 4.12: TF purification. A. TF after 72 hours of 3C cleavage at 4℃. B. Concentration of

TF by Superdex or GST resin. A. Soluble products of cleavage with 3C protease for 72 hours at

4℃, (Wash 1) and subsequent washes (Washes 2 and 3) were analysed. Cleavage products eluting

at a high concentration of glutathione (Elution 1-3) were also analysed. Resin sample is resin after

glutathione elution. B. Soluble protein concentrated by Superdex or GST resin. Concentrated fraction

is TF concentrated protein after a 24 hour 3C digest, further purified via Superdex 200 column and

concentrated. 72 hour 3C digest shows supernatant removed from GST resin after 72 hours with no

further purification steps. Marker sizes (Low-Mass SDS-PAGE marker) are as indicated on the left.

85

4.6 Expression of FFH with Trx tag

FFH with an N-terminal Thirodoxin-His tag was expressed in BL21(DE3) cell line

transformed with the pET(gwA)-32 vector. This was selected as it had the highest

expression and solubility. The cells were grown (§2.8.1), prepared (§2.8.2) and applied

to the Talon resin (§2.8.4). As shown in Figure 4.13, induction of FFH was successful

with a large amount of soluble protein (Clarified lysate lane). However, a large pro-

portion of protein appears not to have bound to the resin, as can be seen in the Flow

through lane.

Upon elution from the Talon resin a contaminating protein was seen to run at

approximately 60 kDa (Imidazole wash 1 and 2). This contaminant ran at the same

theoretical size as the FFH cleaved from the Trx tag. One strategy to remove the

contaminant was to cleave FFH wile bound to the Talon resin. This, however, is not

feasible due to 3C cleavage requires a low concentration of the reducing agent DTT.

Upon the addition of DTT to the Talon resin it reduces the Ni2+ rendering it useless for

protein binding. SEC with the resin elution failed to separate the proteins sufficiently

due to their identical size.

The other strategy attempted was to utilise the inherent binding binding properties

of the contaminating protein to remove it and obtain pure FFH (Figure 4.14). Un-

cleaved FFH was taken from the imidazole washes as shown in Figure 4.13 and cleaved

(§ 2.8.4) (Figure 4.14, Uncleaved and Cleaved). This was then clarified and dialysed

(§ 2.8.4), the protein now free of DTT and imidazole was then bound back to regener-

ated Talon resin to remove the contaminating protein and the cleaved thirodoxin-his

tag. After binding and removal of the supernatant containing FFH, a sample of the

resin was taken. The supernatant contained a commensurate amount of FFH at 60

kDa and the resin contained what appears to be the appropriate amount of Trx-His

tag cleavage product.

86

96

66

42

3222

Size

in k

Da

14

Mar

ker

Unin

duce

dIn

duce

d

Inso

lubl

eCl

arifi

ed L

ysate

Unbo

und

befo

re cl

eava

geIm

idaz

ole w

ash

1

Lyse

d Ce

lls

Imid

azol

e was

h 2

Was

h 1

Was

h 2

Was

h 3

Figure 4.13: Analysis of the expression and purification of FFH. Uninduced, induced and lysate

indicates whole cells treated as described (§2.8.1 and §2.8.2). Clarified lysate and Unbound before

cleavage indicate protein solution before and after binding to the Talon resin. Wash 1-3 relate to

washes of the Talon resin with PBS after protein solution binding. Imidazole wash 1 and 2 relate to

elution of bound proteins from the Talon resin with Imidazole. Marker sizes (Low-Mass SDS-PAGE

marker) are as indicated on the left.

87

Mar

ker

Uncle

aved

Clea

ved

Unbo

und

Imid

azol

e

Unbo

und

wash

1

Post

dialy

sis9666

42

32

22

Size

in k

Da

Inso

lubl

e

| Talon resin |

Figure 4.14: Analysis of the stages of protein purification of FFH. Talon resin purified protein solution

(Uncleaved) was cleaved (Cleaved) and insoluble protein removed (Insoluble). This was dialysed

(Post dialysis) then rebound to the regenerated Talon resin and the supernatant removed (Unbound,

Unbound wash 1). Imidazole shows protein that eluted from Talon resin after washing with Imidazole.

Marker sizes (Low-Mass SDS-PAGE marker) are as indicated on the left.

88

4.7 N-terminal sequencing

N-terminal sequencing was done as described in § 2.9.6 to confirm the previously pu-

rified proteins were those desired. A summary of the results and a comparison to

the sequence determined from Cyanobase is shown in Tables 4.1 and 4.2. The first

2 residues (G and P) are the remainder of the 3C protease cleavage domain, this is

then followed by the start amino acid Met. Sequences showed unambiguously that the

major component in each case is the intended protein.

In Tables 4.1 and 4.2, columns First, Second and Third show the most, second

most and third most abundant residues at each step of the N-terminal sequencing

process. The predicted column indicates the predicted amino acid sequence. The Match

column indicates the similarity between the predicted residue and the experimentally

determined residue. The symbol +++ denotes an exact match.

Table 4.1: TF N-terminal sequencing

Call

First Second Third Match Predicted

G S R +++ G

P D E +++ P

M Y V +++ M

K L E +++ K

V H A +++ V

Table 4.2: FFH N-terminal sequencing

Call

First Second Third Match Predicted

G Y Q +++ G

P R S +++ P

M T N +++ M

F N Y +++ F

D I G +++ D

A M E +++ A

L K Q +++ L

A N P +++ A

89

4.8 Analysis of Antibodies

Test Bleed Dot blot

Test bleeds were done to determine the reactivity of the antibodies over the duration of

the immunisation. These were set up for each rabbit that was immunised with either

TF or FFH. The blots were done at varying concentrations of antibody to determine

its sensitivity and specificity for the desired protein. Processing of the membrane with

the sample of test proteins and cell extracts were treated as described in § 2.9.3 of the

Materials and Methods. Once the rabbits had been immunized with the 3rd booster

and an appropriate period of time waited for the effect of this booster to have taken

place it was determined that the immunity would not develop any more against the

presented antigen.

4.8.1 Analysis of TF and FFH antibodies

Protein concentrations and antibody concentrations as determined from the previous

dot blots were used to analyze the samples via SDS-PAGE and then Western Analysis

(Figures 4.15 and 4.16). This showed good reactivity for the anti-TF antibody easily

detecting 0.01 µg of purified TF. It also, importantly failed to recognise any protein in

the E. coli lysate or the Synechocystis sp. PCC. 6803 TFSpec strain, but did recognise

a band corresponding to the predicted size of TF in the Synechocystis sp. PCC. 6803

WT whole cell lysate.

The FFH antibody did not perform as well with a prominent but weak band at the

predicted size for FFH when examining 1 µg of protein. It also detected proteins in

the E. coli cell lysate lane while detecting noting in the Synechocystis sp. PCC. 6803

WT cell lysate lane. Due to the lack of sensitivity of the antibody this was not used

in any further experiments.

90

Mar

ker

Syn.

-TF

E. co

li ly

sis

Syn W

t

198

83

40

31

Size

in k

Da

0.01

ug T

F0.

1ug T

F

66

131

Figure 4.15: Analysis of TF antibody. Syn. -TF, Syn WT and E. coli lysis lanes relate to 10 µL of

lysed Synechocystis sp. PCC. 6803 TFSpec, Synechocystis sp. PCC. 6803 and E. coli cells respectively,

with an OD600 of ≈ 0.6. TF lanes relate to either 0.01 µg or 0.1 µg TF protein purified as outlined

previously. Marker is Kaleidoscope marker with the sizes indicated on the left.

202

71

31

18

42

133

Mar

ker

E. co

li ly

sis

Syn W

t

1ug

FFH

10ug

FFH

Size

in k

Da

Figure 4.16: Analysis of FFH antibody. Syn WT and E. coli lysis lanes relate to 10 µL of lysed

Synechocystis sp. PCC. 6803 and E. coli cells respectively, with an OD600 of ≈ 0.6. FFH lanes relate

to either 1 µg or 10 µg FFH protein purified as outlined previously. Marker is Kaleidoscope marker

with the sizes indicated on the left.

91

4.9 TF interaction with D1

4.9.1 Ribosomal preparation

Cells were grown under standard light conditions and then exposed to a high light

environment for 30 minutes. To evaluate the association of TF with ribosomes during

damage to D1, ribosomes were prepared under three different light conditions. Thirty

minutes of high light (1000 µE m−2s−1) was chosen from as the photoinhibition exper-

iments this was the time wherein the cells were most photoinhibited. The cells were

then divided and subsequently treated under three different conditions. One aliquot

remained in high light, one was returned to normal growth light (25 µE m−2s−1), the

other kept in the dark. After this recovery period cells were treated with chloram-

phenicol to prevent protein protein synthesis. The dark treated sample was processed

as much as feasible in dark or low red light conditions. Each sample yielded four frac-

tions, pellet fractions or a soluble fractions from the membrane fraction and the soluble

protein fraction for each of the three different cell light treatment conditions. These

were then analysed on a 12% acrylamide gel.

4.9.2 Detection of D1 using FFH and TF

Samples of ribosome preparation were then run a 12% acrylamide gel and transferred

to a nitrocellulose membrane then probed with the Synechocystis sp. PCC. 6803 anti-

TF antibody and an identical membrane probed with an anti-D1 antibody. Figure 4.17

indicates that TF is associated with ribosomes differently in different light conditions.

In the Light condition most TF was detected in the Pellet fraction from the membrane.

This fraction is representative of ribosomes that were pelleted from the membrane

fraction, indicating TF associated with the ribosome attached to the membrane under

the high light conditions. Also in this lane, and the other Light treated Pellet lane, is

a spread of distinct protein fragments from the expected size of TF of 66 kDa to about

33 kDa. This pattern was also seen in the purified TF (Figure 4.15). Under Normal

light conditions, despite the experiment being repeated, there was no detection of TF

in the cells treated under normal light conditions. However, in Dark treated cells TF

92

is detected again, largely in the opposite fractions than detected in the Light treated

condition. The fraction that most signal is detected in is the Soluble sample from

the cytosol, indicating in this situation TF is largely unassociated with membranes or

ribosomes. This experiment was run in an independent duplicate with identical results

in both instances.

Figure 4.17: Analysis of a ribosome preparation from Synechocystis cells prepared grown in high

light, normal light or dark. Lanes are labeled as to the origin of their preparation. Light is high

light, normal is normal light and dark is cells incubated in the dark. All cells were lysed as outlined

in §2.8.7. Cyt refers to the cellular fraction corresponding to the cytosol fraction, Mem relates to

the cellular fraction derived from the Membrane fraction of the first purification stages. Pellet refers

a sample resuspended from the 270, 000 g centrifugation step, soluble relates to the soluble fraction

after centrifugation. Shown on the left are weights of the Kaleidoscope marker.

The same western was done with a D1 antibody to detect if D1 was pulled down in

the ribosome preparation. The experiment was repeated several times but there was

no evidence of D1 in the samples despite controls showing the antibody was working

in the correct manner. This drew a halt to the pull down aim of the experiments as to

93

detect TF associated with D1 successful detection of D1 was required.

94

Chapter 5

Biophysical Characterisation of

Synechocystis sp. PCC 6803 Trigger

Factor

As well as analysing the association of TF with ribosomes, I also characterised the

oligomeric state and apparent secondary structure of TF. In addition to this, crystalli-

sation was attempted.

5.1 Dynamic Light Scattering

This technique uses the temporal fluctuation of light scattering to evaluate hydrody-

namic radii of macromolecules. Because the amplitude of the signal from a particular

species is proportional to both its concentration and the square of its molecular weight,

very large species contribute substantially to the signal, even if present in low amounts.

Samples were analysed as described in the method of § 2.11.1 (Table 5.1). Three prod-

ucts were observed, the first and major oligomeric species with 99.4% of the mass in the

sample had a molecular mass of 101 kDa. Two species of far larger molecular weight

were detected but represent a much lower percentage of the total mass in the sample.

Greater than 99% of the sample is a single species of protein of 101 kDa consistent

with a dimer of TF. Headings of Table 5.1 used to determine the parameters of the

95

individual mass peaks used to fit the scattering data are set out as follows; Radius -

hydrodynamic mass, %Pd - polydispersity, MW-R - Molecular Weight of the protein

per radius, % Intensity - percentage of run signal is in the peak, and % is the percentage

of mass fit to the corresponding peak.

Table 5.1: Dynamic Light Scattering

Peak name Radius (nm) % Pd MW-R (kDa) % Intensity % Mass

TF dimer 4.3 13.6 101 50.9 99.4

Aggregate 1 17.2 13.3 2608 19.5 0.6

Aggregate 2 70.7 13.8 71596 25.9 0

5.2 Circular Dichroism

Circular Dichroism detects the differential adsorption of left and right circularly po-

larised light. This can be used to determine the 2° structure of macromolecules due to

the different inherent CD spectral signatures of both α-helix and β-sheet in a protein.

This in turn can be used to calculate the proportion of the different 2° structures of

the protein of interest. Purified TF protein (§ 4.5) was diluted to 0.33 mg.mL−1 for

initial spectra. Huge variability of the signal in the far-UV spectra suggested that the

concentration was too high. Serial dilution yielded an improved spectrum down to a

concentration of 20 µg.mL−1. This protein concentration was used for the remainder

of the CD experiments.

5.2.1 Secondary Structure proportions

To estimate 2° structure of TF, the sample chamber was held at a constant 20℃ while

5 repeats of the scan from 190 nm to 260 nm was done at a resolution of 2 nm (§ 2.11.2,

spectra as shown in Figure 5.1). There are two minima, at 208 nm and 222 nm. This

is emblematic of an appreciable amount of α helix. Complimentary spectra were taken

at 70℃. Proportions of the 2° structures were using the web based CD data analysis

software Dichroweb as shown below in § 5.2.2, Table 5.2 and Table 5.3.

96

-40

-30

-20

-10

0

10

20

30

190 194 198 202 206 210 214 218 222 226 230 234 238 242 246 250 254 258 262

Wavelength (nm)

Mil

lid

eg

rees

of

Ell

ipti

city

TF trace

Figure 5.1: Circular dichroism spectrum analysis of TF taken at 20℃. An average of 5 scans which

were taken sequentially and averaged. Error bars show one standard deviation of the mean.

97

5.2.2 Dichroweb analysis

Data was submitted to the Dichroweb website as outlined in § 2.11.2. The data returned

is shown in Tables 5.2 and 5.3. Table 5.2 shows there is a decrease in the number of α-

helix segments per 100 aa and also a decrease in the average α-helix length per segment

whereas the numbers of β-strands per 100 aa was scarcely changed and nor had the

average strand length. Table 5.3 showed that the total amount of structured α-helix

(α-helix 1) decreased from a total of about 45% to about 25% (and an increase in the

amount of disordered α-helix (α-helix 2) where as the amount of beta sheet barely

changed. This is also reflected in the decrease of the CD spectral signature of α-helix

seen in the 70℃ trace of Figure 5.3. Interestingly the amount of α-helix seen in the

70℃ is about the same determined from the Vibro cholerae crystal structure.

Table 5.2: Dichroweb analysis: Comparisons

Variable 20℃ 70℃ V. cholerae prediction

NRMSD 0.000 0.000 -

Helix segments per 100 residues: 2.6 2.5 2.6

Ave helix length per segment: 21 17 16

Strand segments per 100 residues: 4.0 3.9 2.9

Ave strand length per segment: 4.4 4.7 7.4

Table 5.3: Dichroweb analysis: Secondary structure proportions

Temperature Helix 1 Helix 2 Strand 1 Strand 2 Turns Unordered

20℃ 0.44 0.10 0.10 0.08 0.06 0.22

70℃ 0.27 0.15 0.11 0.08 0.05 0.34

5.2.3 TF thermal denaturation

TF was exposed to a range of temperatures to analyse the response of the protein to

an elevated temperature and to analyse its thermodynamic stability. This was done

using a identical sample of protein. The protein was cooled to 5℃ for 30 minutes prior

to the start of the run to allow equilibration. Upon commencement of the analysis

98

the temperature was increased in 5℃ increments (§ 2.11.2), the temperature allowed

to stabilize for 30 seconds after reaching the temperature before measurement and

adjustment to the next temperature. A 3-dimensional plot of the resulting data way

outlining its stability (Figure 5.2) show a marked reduction in the rotation of the light

in the central wavelengths (236 nm to 296 nm). Spectra from several key temperatures

that indicate partial recovery of structure with decreasing the temperature and the

partial recovery are shown in Figure 5.3. The plateau in further spectral change above

45℃ indicates that the change is complete, no further interruptions to the 2° structure

of TF were seen in the temperatures examined.

196

200

204

208

212

216

220

224

228

232

236

240

244

248

252

256

260

5°C

20°C

35°C

50°C

65°C

60°C recovery

45°C recovery

30°C recovery

15°C recovery

-40

-30

-20

-10

0

10

20

30

Millidegrees of Ellipticity

Wavelength (nm)

Temperature (C°)

20-3010-200-10-10-0-20--10-30--20-40--30

Figure 5.2: CD spectra of TF thermal denaturation from 5℃ to 70℃ and recovery. Shown is the

change in the rotation of the polarised light at various wavelengths (nm) with changing temperature

(℃) Colour indicates 10 Millidegree ellipticity ranges. Grid is 2° by 5℃.

99

-40

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-10

0

10

20

30

190

192

194

196

198

200

202

204

206

208

210

212

214

216

218

220

222

224

226

228

230

232

234

236

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240

242

244

246

248

250

252

254

256

258

260

Wavelength (nm)

Mil

lid

eg

rees

of

Ell

ipti

city

5°C20°C70°C20°C recovery

Figure 5.3: CD spectra of TF at key thermal points. Spectra at 5℃, 20℃, 70℃ and 20℃ recovery

during a gradual increase in temperature and then 20℃ once the sample had been cooled.

100

5.2.4 Melting point of TF

This high quality sampling allowed the determination of the Tm from data at two

wavelengths, 208 nm and 222 nm as shown in Figure 5.4. Figure 5.4 was plotted with

a polynomial line of best fit and line inserted to illustrate the Tm (℃) as half of the

difference between the maximum and minimum values of these two wavelengths. The

208 nm wavelength data gave a Tm value of 37℃ and the 222 nm data gave a Tm

36℃.

-40

-35

-30

-25

-20

-15

-10

15 20 25 30 35 40 45 50 55

Tm of TF

222nm

208nm

Mola

r Elip

ticit

y x

10

-3 (

deg.c

m2.d

mol-1

)

Temperature (°C)

Figure 5.4: Cross-section of the CD spectra of TF during thermal denaturation measured at 208 and

222 nm, the black lines indicate the points that mark half the difference between the maximum and

minimum values, this leads to the Tm of TF on the X-axis.

101

5.3 Crystal Trays

Circular Dichroism data and Dynamic Light Scattering data suggested that the TF

protein as purified may be a good candidate for crystallisation. Trials were set up

using the Hampton Research Crystal Screen HT and 2HT (§ 2.11.3). Trigger Factor

from the same batch as the CD and DLS experiments was concentrated to 9 mg.mg−1

and was applied to the sample wells of Mosquito Robot. Hanging drops of 0.2 µL were

created over the wells of buffer. This was done in duplicate, one of which was stored

at 4℃ and the other was stored at 18℃. These were examined immediately and notes

of each condition taken then examined at regular intervals (§ 2.11.3). Figures 5.5 and

5.6 show typical examples of hanging drops in which produced different results. Figure

5.5 shows two different hanging drops, on the left, the sample had many micro crystals

indicating no problem with initiation of crystal growth and the right hanging drop

shows a single small crystal that failed to develop any larger. Figure 5.6 shows two

different variables that failed to develop crystal but developed particulate precipitate

or an oily precipitate. Tables showing results from all conditions are shown in the

appendices.

Figure 5.5: Hanging drops examined under magnification showing small crystals.

102

Figure 5.6: Hanging drops examined under magnification showing typical precipitate.

103

Chapter 6

Discussion

6.1 Creating the Synechocystis sp. PCC 6803 TFSpec

mutant

The Synechocystis genome readily undergoes homologous recombination with a plasmid

construct containing Synechocystis DNA sequences. Synechocystis sp. PCC 6803 was

transformed with a gene construct comprising the up and downstream regions of the

TF gene with an antibiotic-resistance cassette causing an interruption in the ORF

(TFSpec).

Creating the construct for the interruption knockout was done easily due to the

PCR conditions already having been determined by previous work. Once the PCR

product was obtained, the ligation of the PCR product into pGEM-t yielded several

clones that displayed the desired restriction digest pattern. Insertion of the spectino-

mycin resistance sequence into the XmaI restriction site yielded one clone that was

confirmed by restriction digest and sequencing (shown in the appendix) of the inserted

region of the putative clone. This was then used for the transformation of Synechocystis.

Synechocystis carries multiple genome copies per cell, hence to obtain a pure mutant

phenotype, all WT genome copies need to be replaced. The cells are therefore grown

under antibiotic selection for a period of 4 to 8 weeks. If the gene is essential, the

segregation may never be complete, as to maintain its viability each cell must retain

105

the essential gene whilst also maintaining the antibiotic selection.

Transformants were analyzed weekly for segregation via PCR. This is a convenient

and rapid method to screen for segregation of WT and TFSpec genotypes as only a

small number of cells are required - as few as a colony of the cells can be used as a

template for PCR amplification. After about 6 weeks PCR indicated that the segrega-

tion/replacement of the WT genome was complete. PCR can be a selective technique,

not amplifying all species present, so a negative result is not always definitive. There-

fore, a Southern blot was done to compare and confirm that there was no WT genome

remaining. This was conclusive that all copies of TF in the genome had the desired

insertion and were now TFSpec. This also shows that TF is not an essential gene in

Synechocystis, in line with the findings of others for E. coli (Kandror and Goldberg

(1997)).

6.2 Phenotype of the Synechocystis sp. PCC 6803

TFSpec knockout

6.2.1 Photoautotrophic growth curves

Growth rate of the Synechocystis TFSpec strain was measurably lower than the WT

strain. This could be attributed partially to a decrease in the total cellular function

of the cells due to not having an important part of the protein translation and folding

pathway. It could also be attributed to a decreased rate of repair during the natural

cycle of photoinhibition. The difference between the TFSpec and WT was predicted

to have become more severe at lower temperatures however this effect was not clearly

seen (Figure 3.7). This could be due to several reasons, firstly, the temperature chosen

from may have been too cold with both strains reaching a plateau of not being able

to perform adequately. Alternatively, the effect was not seen over the duration of the

experiment as the cold-slowed cellular doubling time meant a meaningful difference

was not yet apparent. This could also indicate that the role of TF, and therefore the

TFSpec phenotype, may differ in E. coli and Synechocystis.

106

Future experiments may investigate the cells when grown in a variable light inten-

sity, higher light may the slow the growth. This decrease in growth would likely be due

to a decrease in the proportion of active vs. photoinactivated PSII rather than a total

decrease in the total amount of PSII. This decrease in active centres would accumulate

over time as they got damaged and the repair could not take place at the rate that the

centres were being inactivated.

The photoautotrophic growth curves show that TFSpec cells grew slower than their

WT counterparts. However, the cells were grown in light levels that are considered low

and unlikely to stress the cells. To further investigate the photosynthetic capabilities

of TFSpec cells, experiments were done by investigating the rate at which the TFSpec

evolved oxygen compared to WT. These experiments directly examine the photosys-

tems and their ability to function in a relatively high light condition.

6.2.2 O2 evolution

The TFSpec cells displayed a decreased O2 evolution compared to WT, normalised a

base level of 1 as shown in Figure 3.8. This is most likely due to lower amounts of func-

tional PS II as a consequence of having an interrupted protein chaperone and folding

pathway. The inflection point of the O2 evolution in the TFSpec cells at approximately

30 seconds after the start of illumination is an interesting feature which could be due

to the active photosystems being inhibited faster than the normal rate and failing to

be recruited back from the inactive form of PS II. This is interesting in that it is clear

that the TFSpec strain has the same initial rate for about 30 seconds, then the clearly

decreased rate for the subsequent testing time. Such a fast response cannot be ex-

plained by either less active or a lower number of PSII centres. It is consistent with

photodamage overwhelming a protection or repair mechanism. Photoinhibition studies

could illuminate further interactions of this nature.

107

6.2.3 Photoinhibition studies

Photoinhibition of WT versus the TF knockout was examined to determine if there

was any discernible difference in the recovery of the knockout after exposure to high

light levels. A change in the pattern seen could be then implicate TF in the recovery

mechanism. The cells had a very similar response while they were being exposed to the

1000 µE m−2s−1 light and that they both adapted to this level of light after a brief drop

in the O2 output. This changed in the recovery phase showing that the WT continued

recovering to overcompensate, in a lag type response to the potential for decreased

photosynthetic output. This compares to the TFSpec strain which achieved the same

levels two hours after the WT peak. The peak was also elongated, implying that the

PS II of the TFSpec strain was still fully functional but other repair mechanisms were

slower at dealing to the light challenge.

6.2.4 E. coli phenotype comparison

E. coli growth experiments with WT and ∆tig (not shown) repeated the cold phenotype

seen by Kandror and Goldberg (1997) using an independently constructed ∆tig strain.

The levels of colony survival were almost identical as those reported in their paper

with almost no survivors after 6 days incubation at 4℃. This finding had not been

replicated in literature since its initial report.

Future experiments could examine if this phenotype is repeatable in Synechocystis

TFSpec. Synechocystis has a narrow temperature range and room temperature can be

considered cold (Allakhverdiev and Murata (2004)). Therefore, a dramatic difference

between WT and the TFSpec may be seen when grown at 4℃ as E. coli ∆tig colonies

were decimated after 6 days of growth. However, no difference was seen for growth at

18℃. Further to this, the Synechocystis TFSpec cells may display another phenotype

seen in E. coli and fail to split on cell division as reliably as WT. Hence, filamentous

strands of the normally isolated cells may be seen.

108

6.3 Truncations of TF

TF has three different domains, 1, 2 and 3 these are the ribosome binding domain, a

PPIase domain and a central domain chaperone domain. As there was a reproducible

∆tig (TFSpec) phenotype in Synechocystis it could be used to analyse the functions of

the different domains through creating deletions of the domains in different combina-

tions.

Figure 6.1: TF domain boundaries. TF protein sequences from Vibro cholerae and Synechocystis were

aligned using the clustalw multiple alignment algorithm and the domain boundaries were overlaid from

the Vibro cholerae. Shown in blue is domain 1 with the gold de-marking the ribosome binding motif,

green is the domain 2, the chaperone domain and magenta is domain 3, the PPIase domain.

Domain 3 is a spatially unified domain but its sequence is interrupted by domain

2. Domain 1 is at the 5’ end of domain 3 (Figure 6.1). To design a way for the trun-

cations to be made, restriction enzyme cleavage sequences with identical four central

nucleotides were identified and designed into the central region of PCR primers span-

ning each of the domains boundaries. This would allow the different combinations that

were to be created. Truncations of interest were: Domain 1, the ribosome binding do-

main by itself which could confirm that it is sufficient to bind the ribosome and also test

109

whether it has any nascent-chain binding effect. Domains 1 and 2 to further examine

the chaperone ability of TF and domains 1 and 3 to examine the PPIase functionality

of TF.

This method had the potential advantage of generating all of the separate compo-

nents sequentially and potentially needing only six PCR reactions. The initial method

to amplify the fragments directly from the genomic DNA led to numerous unknown

bands. The plasmid generated for the interruption of TF with the spectinomycin resis-

tance gene could not be used as it lacked sufficient flanking region outside of the gene

to support recombination. PCR was then done on the genomic DNA using the new

primers to amplify the flanking regions of the gene. This led to a large single PCR

product that was easily discernible and able to be purified. Insertion into pGEM-5

then allowed the amplification of the first segment.

This was relatively straight forward as it was a simple PCR, this product was used

in the subsequent PCR as the forward primer. Determining the optimum conditions for

the amplification using this mega-primer required a systematic combing of the different

combinations and permutations of template, primer, and salt concentrations as well as

temperature and incubation times. However, despite best efforts to reliably repeat

this reaction, it proved very temperamental. Low yields of the product, the 1.9 kb

Ac-g primer, and the unpredictable nature of obtaining it and the further evasiveness

of conditions that would enable the amplification of the last product using Ac-g as a

primer led to this approach being abandoned.

The other approach to obtain a deletion construct to was to design another primer

complimentary to the reverse primer that overlaps the C-terminal domain (Primer G).

Then attempt to ligate the pGEM-5 vector, N-terminal flanking region, chlorampheni-

col resistance gene and the C-terminal flanking region. The isolation of the different

regions to be ligated was straight forward. When ligating due to there being multiple

inserts the molar ratio of fragments used was 1:1:1:1. This was repeated several times

and the few colonies that grew were analyzed by restriction digest. The restriction

analysis showed that there was one colony that appeared to have the correct digest

pattern. This was sent for sequencing and it showed that the N-terminal flanking re-

110

gion was missing and that the restriction site that the construct was thought to be

cleaved at wasn’t there. This was tried multiple times however no colonies were found

that exhibited the correct restriction digest pattern. Due to lack of results and time

constraints this line of work was put on hold to be continued in a further project.

6.4 Analysis of non-Synechocystis sp. PCC 6803 anti-

TF antibodies

6.4.1 Cross reactivity of antibodies

Phylogenetic analysis

Cross-reactivity of the anti-TF antibodies was not predicted by phylogenetic analysis

(Figure 4.1). There are some regions of 4 or 5 amino acids that show similarity through

all species except A. aeolicus. The A. aeolicus sequence looks by far the most different

from the others and has been sorted as the outlier, while E. coli and H. influenzae look

the most similar. It is also worth noting that Synechocystis, as a species, is dissimilar

to the others.

Western analysis of various TF antibodies

As predicted by phylogenetic analysis, cross-reactivity of anti-TF antibodies was not

seen. This is likely due to the dissimilarities noted in the phylogenetic analysis with

few identical sequence similarities. Although some cross-reactivity was detected, none

of the antibodies detected a protein corresponding to the correct size of TF. The bands

detected, especially given the very large, or small, molecular weights detected are likely

the antibodies spuriously binding random proteins.

111

6.5 Expression of TF and FFH

6.5.1 Gateway construction

Primers were created to amplify FFH and TF. They were designed to be complemen-

tary to at least 18 nucleotides. The 5’ end of the forward primer encoded a protease

site at the N-terminal of the translated protein and included a XmnI restriction digest

site. The reverse primer included a XhoI restriction digest site. The genes were in-

serted into the Gateway pENTR-11 vector as outlined in the results Figure 4.4, this

was achieved with relative ease compared to the previous efforts of isolating the genes

themselves. Recombination and transformation of the respective pENTR-11 vectors

into the destination vectors was similarly straight forward. Results (Figure 4.5) illus-

trate that over the ten transformations done, 8 of them were successful and show the

expected restriction digest. The proportion of successful colonies is seemingly helped

by the double selection criteria of the antibiotic resistance and the lethal ccdB gene.

6.5.2 Trial Expression

The pDEST vectors were transformed into the different cell lines for expression as out-

lined in the results chapter. The BL21(DE3) cell line transformed with the pDEST-

15 vector was best for the expression of TF and BL21(DE3) cells expressing the

pET(gwA)-32 best for FFH. Expression was monitored and objectively compared by

eye to determine the greatest and most soluble expression. Samples were run in dupli-

cate to determine consistency between the expression levels within the cell line. There

was a large amount of variation seen in the expression levels of different vectors. No

pattern was could be determined for predicting if the recombinant proteins would ex-

press well from each vector, thus, using the GateWay expression system was found to

be valuable to easily clone and select the optimum constructs.

6.5.3 Full scale expression of TF and FFH

TF expression worked well, the process for purification of TF was followed as set out in

the materials and methods. This initially consisted of expressing the protein, lysing the

112

cells, centrifugation of the cell lysate. The supernatant that remained after centrifu-

gation was the subjected to the GST resin purification which as noted in the methods

was done in the batch method. It was assumed that after this the protein would still

not be of sufficient purity to proceed and that it would need further purification via

Size Exclusion Chromatography. This was done (See appendix), but it appeared that

the protein behaved poorly in the column, and led to a large proportion of the protein

appearing tightly concentrated at the very end of the run. However, upon SDS-PAGE

analysis this appeared to run at the correct size for TF. This result was perplexing as

appearing in the UV chromatogram during the analysis of the column run indicated

misbehaving protein. Protein eluting from the column at the same volume as salt

would indicate that the protein was retarded by potentially sticking to the column.

Protein that behaves poorly in the inert SEC column was deemed to have a flaw and

may indicate that it lacked the native conformation. Therefore other avenues of purifi-

cation were pursued. Further analysis of the GST resin and a sample of the remaining

supernatant in the resin showed that there was a much larger amount of cleavage of the

protein after the extended cleavage time being incubated at 4℃. In addition, protein

obtained was of a equal, if not superior, purity than the samples eluting at the correct

the theoretical molecular mass of TF on the SEC column.

TF took a long time to digest with 3C. Although this is potentially negative for

the stability of the protein it was seen that the protein maintained a uniform structure

in various biophysical characterisation tests carried out later on. Long 3C digestion

times for other proteins are also observed so this result was not either unexpected or

unusual (Willbanks, Personal Communication). The long digestion was also off set

by the relatively high purity that was obtained using the single purification method.

The long time for the purification was therefore compensated by the fact that further

purification was not necessary.

FFH purification proved more troublesome. This was due to a protein of identical

size as the cleaved FFH eluting from the resin when washed with imidazole. FFH-fusion

could not be cleaved while still bound to the Talon resin as the reductant required by

the 3C protease would reduce the Ni2+ in the resin effectively destroying it. Therefore

113

it was important to elute the bound proteins with imidazole to cleave the Trx-His-FFH

tagged protein while separate from the resin. Dialysis was then used to remove both

the DTT and the imidazole, and the sample equilibrated back to standard buffer. This

was then able to be run against the resin again which removed the cleaved tag and the

contaminating protein, leaving FFH in the supernatant. This approach was verified

by then eluting the proteins from the resin with imidazole to determine what species

were bound confirming only a minority of uncleaved fusion protein, the contaminating

protein and the TRx-His tag. This approach yielded ample protein of a high purity for

the further steps of immunising the rabbits against FFH. The contaminating protein,

through experience of others, appears to be the molecular chaperone dnaK which will

bind to a variety of different compounds. Once removed, it did not represent a further

problem

6.5.4 N-terminal sequencing

Small amounts of the TF and FFH were transferred to individual membranes and sup-

plied to the Protein Microchemistry Facility. This was then analyzed and significantly

strong signals were detected for the predicted sequences as determined from the ge-

nomic sequence both from the published sequence and the DNA sequencing done on

the pENTR-11 vectors. This was another confirmation indicating that the protein was

reasonably pure and that it was in fact, the correct protein.

6.6 Analysis of anti-TF and anti-FFH antibodies

The test bleed dot blots were done immediately following the test bleed. This was

done to determine the progression of the immune response of the rabbits to the antigen

being presented. Although two rabbits were immunised for each antigen, both rabbits

immunised against FFH failed to provide titers of antigen that were sensitive enough

to detect FFH in cells. It also detected cross-reactive proteins from E. coli. The

rabbits immunised against TF however, had a much more substantial response to the

TF antigen and provided anti-TF antibodies that were able to detect TF at low levels

114

and with a high dilution of antibody (1:100,000).

TF was detected at approximately 0.5 ng in whole lysed Synechocystis and not

detected in the Synechocystis TFSpec strain or any cross-reactivity detected in the E.

coli control (Figure 4.15). A large number of bands was seen in the TF protein lanes,

this was attributed to TF break down products. However, it is unclear how the protein

was cleaved into such distinct bands and in such high proportions. The protein, when

purified was pure and protease activity prevented with a cocktail of inhibitors therefore

is not anticipated to be proteolytic degradation.

6.7 Ribosome associated TF and FFH

6.7.1 Ribosomal preparation

The ribosomal preparation protocol was amended slightly over various iterations to find

optimal centrifugation times to pellet ribosomes. Coomassie staining (data not shown)

showed the characteristic ribosomal subunit pattern, this was ascertained by compar-

ison to an E. coli ribosomal subunit pattern run on an SDS-PAGE gel (Wilbanks,

personal communication). In the comparative E. coli preparation the TF protein was

detected in the ribosomal fraction, as it was pulled down attached to the L23 subunit.

6.7.2 Detection of FFH and TF bound to membrane associ-

ated ribosomes

The detection of TF in the ribosomal preparation provided several unexpected results.

Firstly is the bands that are visible in the different samples, the two bands detectable

in the Light grown cytosolic pellet fraction and the 4 distinct bands in the Light grown

membrane pellet fraction. These are not found in any other lanes. These same bands

appear in the analysis of purified TF that was run in analysis of the TF antibody

and is thought to be some break down product of TF but is unclear why it is only

detected in Light grown cells. Also interestingly is the pattern of ribosome associated

TF detected between the different light treatment conditions. Patzelt et al. (2002)

115

measured amounts of ribosomes and TF in E. coli cells and found that there was a

ratio of about two TF proteins to each ribosome, half being soluble in cytosol and

just about every ribosome having TF associated. However, this finding indicates that

TF-ribosome binding may be a very dynamic situation with different growth or physi-

ological conditions showing varying association with ribosomes and membranes.

The presence of TF in the samples was promising for the continuation of the pull

down experiments. However the D1 antibody did not detect any D1 protein in the

ribosome samples. There are several possible explanations for the lack of detection.

One is that there was no D1 in the sample. The other is that the D1 antibody was not

sensitive to detect only the N-terminus of the protein.

There is evidence that this protocol specifically extracted ribosomes as intended.

There are bands in the preparation from the pellet sections that correlate with the pres-

ence of TF. The presence of a small protein such as TF in the sample indicated that it

was pelleted with a far larger species such as a ribosome. This, therefore, indicates that

there is likely to be ribosomes in the sample. Previous studies (Tyystjarvi et al. (2001))

have shown that D1 can be isolated from bound ribosomes that had stalled translation

at 17 kDa after exposure to high light conditions followed by darkness. Hence the cells

that were placed under high light and then placed into darkness while the ribosomes

were isolated should posses a large number of partially translated D1 proteins. D1,

truncated or full length was not detected after repetition of the experiment. It seems

likely that the antibody was not sensitive enough for the small amount or length of

the protein. An antibody specific for the N-terminus of the protein is commercially

available and is recommended for future investigations.

116

6.8 Biophysical Characterisation

6.8.1 Dynamic Light Scattering

Dynamic Light Scattering is a valuable tool for assisting in determination of suitability

of a protein sample for crystallisation. It allows the user to determine the homogeneity

of their sample via a monochromatic light beam. The change in the motion of the

light when it strikes the particles in the solution causes a change in the wavelength of

the light, this change is directly related to the size of the particle, and therefore it is

a very good tool for determining the species of protein in a solution (Sartor (2009)).

Oligomeric states and homogeneity are important variables for the crystallisation of

proteins. DLS allows the measurement of the polydispersity and the amount of ag-

gregate in the sample. Polydispersity is determined by the relative standard deviation

of the sample and infers the mass distribution characteristics of the sample. This can

be divided into three groups, less than 20% (low), from 20% to 30% (medium) and

greater than 30%(high). A low Pd indicates that the sample is a suitable candidate

for crystallisation (Doublie (2007)). As seen there were three species detected, one of

which was the majority of the sample analysed with a total of 99.4% of the total mass

present. This was encouraging the sample is in a single state. The mass, however

did not correlate with the mass for a single TF protein on its own. This led to the

conclusion that it exists, at least when purified, in a dimer state as the mass corre-

lates precisely with a TF-dimer. This was an unexpected result but correlated exactly

with the with the finding of Patzelt et al. (2002) in E.coli and the crystal structure of

Ludlam et al. (2004) in V. cholerae.

The mass percentages indicate that TF is in the dimeric state, as TF had been

crystallised in the dimeric configuration this was encouraging that for the preparation to

form crystals. The other valuable information from the results obtained and illustrated

in Table 5.1 is the low percentage of polydispersity (Pd) of the sample. As outlined

above the lower the value of the % Pd the more homogenous it is and the more likely

that the sample is suited to crystallisation.

117

6.8.2 Circular Dichroism

Secondary Structure Proportions

Circular Dichroism was done to determine if the TF protein was in a folded state. This

was another test to determine if it would be suitable for crystallisation trials. To do

this the protein was scanned five times to allow an accurate calculation of the error

in the data. The analysis from Dichroweb indicated that there was a considerable

amount of α-helix, a low amount of β-strands, turns and random loops (As illustrated

in Table 5.3). This was organised to have an average length of helix of about 21 aa

per segment with about 2.6 segments per 100 residues gives approximately 50% overall

α-helical structures. This is compared to the crystal structure of the Vibrio chlorae

TF that has approximately 43% α-helical structure. The β-sheet indicated by CD

indicates that there are many short strands with a total of about 20% of the overall

structure also comparable to known structure that indicates that there are 20% β-sheet

structures. There is a contrast in the length of these sequences with the Vibrio chlorae

TF structure showing fewer strands with longer length compared to Synechocystis TF

with more short β-sheet structures.

It is understandable that there is an under-estimation of the β-sheet structures as

the major differences between β and α CD spectral signatures is seen in the magnitudes

of the negative peaks between 210 and 230 nm and a positive peak at 190 nm. With the

peaks in the α-helical spectrum having approximately 5 times the magnitude of those in

the β-sheet region. Therefore, it is not unexpected that if there is a large amount of α-

helix present then the sheet would be underestimated or at least incorrectly estimated.

TF thermal stability

To analyse the thermal denaturation of α-helical structures the wavelengths of 208

nm and 222 nm are informative. These show the S-shaped curve of Tm and also

indicates that although some of the structure was retained it largely became random

coils. Both wavelengths were interpreted and gave the same temperature of 36℃±1℃.

The temperature was increased from 5℃ to 70℃ and then lowered to 10℃ as shown in

Figure 5.2. This indicated the change in the structure of the protein as the temperature

118

increased, it also demonstrated a limited recovery from the unfolding caused by the

temperature increase. This was, however, not enough recovery to return the protein

to its native structure and it appeared to stabilize and not recover any further from

35℃. Experimental data shows a curiously low Tm but as Synechocystis is adapted

to grow in a tight temperature range this is perhaps not surprising. Further to this,

TF is classed as a cold shock protein (Kandror and Goldberg (1997)) and there is no

evidence that its activity is required at higher temperatures.

6.8.3 Crystallography setup and trials

Optimal conditions for crystal nucleation and growth are difficult to predict and hence

a broad screening method is a fast and efficient way for analysing numerous conditions.

There are many proteins that have been previously crystallized and many have been

crystallised in a select group of buffers and pHs. A commercial crystal screen allows for

the examination of these various conditions in high quality salt, polymer and solvent

solutions. During the screening approximately 600 conditions were analysed for crys-

tals, amongst these there were varying degrees of results. The range was from blank

samples to single small crystals to many small crystals. However, a promising candidate

condition with few small crystals was chosen and a screen varying the PEG molecular

weight and percentage, different divalent cations and pH, ultimately proving fruitless.

Other options were also considered, such as increasing the protein concentration. This

however was not done as many of the hanging drops showed precipitation, evidence

that the protein was already beyond solubility. One further option not examined was

to create a different construct as proved successful for Ludlam et al. (2004).

6.9 Conclusions

The creation of a TFSpec mutant of Synechocystis was successful and proved that TF

is not an essential gene in Synechocystis. This Synechocystis TFSpec strain was used

to then characterise the phenotype of the mutant which demonstrated a measurably

lower photoautotrophic growth curve and decreased O2 evolution compared to WT.

119

It also demonstrated that active photosystems being inhibited faster than the normal

rate which is likely due to a change in the PS II replacement mechanism rather than a

change in the physiology of the cells.

Biophysical characterization of Synechocystis TF by CD examination showed ex-

tensive 2° structure of TF with distinct similarities to the known crystal structure.

However, interestingly results from analysis of TF in different cellular fractions

indicate that TF-ribosome binding may be a very dynamic situation with different

growth or physiological conditions showing varying association with ribosomes and

membranes. Preparation of membrane bound ribosomes from cells grown under high

light showed a much greater level of TF than those from other conditions. This supports

a role for TF in the repair of photodamaged PSII. This conclusion could be strengthened

by detection by an appropriate sensitive antibody.

120

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125

Appendix A

Creation and physiological analysisof Synechocystis sp. PCC 6803TFSpec

A.1 TF in pGEM-t amplified with T7 sequencing

primers

127

TF-pGEMt.T7.ab1 - Avg. Quality: 0.0A:26%, C:24%, G:26%, T:21%, N:41, Total: 1292

- - - - T - - Q - A PA M Y R KW K R W R W C A C C G G W A K G C AG Y C TG C TC

10 20 30

P A A W P R D S N R G M Q MC C G G C C G C C T G G C C G C G G G AT T C TA A T C G G G G C AT G C A G A

30 40 50 60

M G N A V R K Q Y Q A * L SA T G G G C A A C G C A G T C A G A A A G C A G T A T C A A G C T T A G T T A A G

70 80 90 100

S P V G Q S C W G K N Q P KG C C C A G T T G G T C A A A G T T G T T G G G G T A A A A A T C A G C C A A

110 120 130 140

K K K K L I V T Q G I A PA A A A A G A A A A A A C T A A T T G T G A C G C A G G G C A T T G C C C C C

150 160 170 180

P * T A K P L L N C P A R KC T A A A C C G C A A A G C C A T T A T T G A A T T G T C C A G C T A G A A A

190 200 210 220

2009-05-09 23:41:58 +1200 Page 1 of 6www.mekentosj.com


K I I D L V P F S Q T G L GA A A T C A T T G A T T T A G T C C C A T T C T C T C A A A C C G G C C T G G

230 240 250 260

G D P P A G K D S R P R KG G G G A C C C C C C G G C G G G A A A A G A T T C T C G C C C C A G G A A

270 280 290

K K W H P A Y I M I N W V DA A A A A T G G C A C C C G G C T T A T A T T A T G A T C A A T T G G G T T G

300 310 320 330

D R H R S S P S H C V A GG A C C G C C A C A G G T C T T C C C C G A G C C A T T G C G T T G C G G G

340 350 360 370

G L E G K H F P R S K * T *G G C T A G A A G G G A A A C A T T T C C C T C G G T C T A A G T G A A C C T

380 390 400 410

* H N T G R R A H R P I ST A G C A T A A C A C G G G T A G A A G G G C C C A C A G A C C G A T T T C

420 430 440



S A V L P S P P G V N L V PC T G C A G T C C T T C C C A G C C C T C C A G G G G T G A A C T T A G T G C

450 460 470 480

P T A P I V S P S R F P IC C C A C T G C C C C C A T T G T T T C A C C A T C G A G A T T T C C C A T

490 500 510 520

I N E S Y P G K I A R * S ST C A A T G A A A G T T A C C C A G G A A A A A T T G C C A G A T A G T C A A

530 540 550 560

S W A R N * D S R H S V EA G T T G G G C T A G A A A T T G A G A T T C C C G C C A C A G C G T C G A

570 580 590

E K S L * E R R Q K V N P HA A A A A A G T T T A T G A G A A C G T C G T C A A A A A G T T A A C C C G C

600 610 620 630

H R * Y S W V P S G E G AC A C C G T T A A T A T T C C T G G G T T C C G T C G G G G G A A G G T G C C

640 650 660 670



A P G H C Y P A F G T K L YC C C G G G C C A T T G T T A T C C A G C G T T T G G G A C A A A G C T A T A

680 690 700 710

Y Q G H G H * R A D R R Q HA T C A A G G C C A C G G C C A T T G A A G A G C T G A T C G A C G A C A G C A

720 730 740 750

H * S R G * A R R T T H H WA T T A A A GC C G C G G T T A A G C A A G A A G A A C T A C C C A T C A T T

760 770 780

W Q F F P A L G H G K P N ST G G C A A T T T T T C C C T G C G C T C G G A C A T G G A A A A C C T A A T T

790 800 810 820

S N L * P * S A P H H * G GT C A A A T C T T T G A C C C T G A A G C G C C C C T C A C C A T T A A G G T G G

830 840 850 860

G S G C V P R S G I * T G EG C A G C G G A T G T G T T C C C C G A A G C G GA A T A T G A A C C G G A G A

870 880 890 900



E L * K I T A Q A E E I E YA G C T A T A A A A A A T C A C T G C C C A G G C G G AA G A AAT C G A A T A C

910 920 930 940

Y S V D A V D Q W L K G E Q EC A G T G T C G AT G C C G TT G AC C A G T G G C T CAA G G G A G A AC A GG

950 960 970 980

E N G L P L S R W R T V L RGA A A A C G G G C T A C C C T T G T C C C G G T G G A G G AC C G T C C T G C G

990 1000 1010 1020

R P G R S G H C G L - - F QG C CC TG G G A G AT C T GG C C A T TG T G G A C T A C S C S C T T T T C A

1030 1040 1050 1060

Q - Q - M V K L - R R S - NA G K G G CA G A A R AT GG T C AA G C T G G R R A G GC G A TC GY C G A A

1070 1080 1090 1100

N E G S D L - D P T D VA TG AA G G G T C T G A T T T G A G K G A C C CT A C A G A C G TC

1110 1120 1130



V V C G W - - G R N - E WC G T T T G T G G C T G G T W T TG K G G A C G G AA T K T S G AA T G

1140 1150 1160 1170

W R * * N Q - D S G T SG G C G G T G A T G A A A C C A A A C K G A TT CC GG T A C T T C

1180 1190 1200

S E - S - T - R KC G GA G A M WT C C Y T G A C T K G G C G G A A A

1210 1220 1230

K C - D * - D IA T G T K T T G A TT A G YY A G A T A

1240 1250

I G - L M - -A T C GG A C K C C T G A T G G M A K A

1260 1270

- - P - C - --A AC T Y C C C A M A TG T G K A S C G RA

1270 1280 1290


A.2 TF in pGEM-t amplified with SP6 sequencing

primers

135

TF-pGEMt.SP6.ab1 - Avg. Quality: 0.0A:20%, C:26%, G:23%, T:28%, N:41, Total: 1296

- - - - - - - - - - V GA RW S S M M M M S W K K W W Y Y Y W Y R Y W Y TC M M G C G T TG G

10 20 30

G S S P I W S T C R R P H *G G AG C TC T C CCA TA T G G T C G AC C T G C A G G C G G C C G C AC TA G

40 50 60 70

* * L L G R R K G R E N * EG T G AT T G T T A G G T C G AC G G A A A G G T A G G G A A A A T T A A G A A

80 90 100 110

E K G G Q G S S R R P R S GA A A A G G C G G A C A G G G A T C A A G C C G G C G T C C A A G G A G T G G

120 130 140 150

G E K K L F L Y F A L G * LG G G A A A A A A A A C T T T T C C T T T A C T T T G C T T T A G G T T A A C T

160 170 180 190

L N F V A D M V S S Y L DT C A A C T T T G T T G C C G A C A T G G T C T C A T C T T A C C T A G A T

190 200 210 220



D L L E P W G T N A L G Q GT T T A C T T G A G C C T T G G G G G A C T A A C G C C C T A G G A C A G G G

230 240 250 260

G G Q E V N L P C Q F T YG A G G G C A G G A G G T T A A T C T G C C C T G T C A A T T T A C C T A T

270 280 290 300

Y S G D F S S S L R R P S MT T C C G G A G A T T T C T C C T C T A G T T T G C G G A G G C C C A G T A T

310 320 330 340

M P L T P Q G * F G P T GT G C C T C T G A C A C C C C A A G G G T A A T T T G G G C C A A C G G G T

340 350 360 370

G Y S S S E V A S T S T M AT T A C T C T T C G T C G G A G G T T G C T T C C A C A T C C A C G A T G G C

380 390 400 410

A S S S A S S S G V S S SC T T C T T C C T C T G C A T C G T C G T C G G G A G T A T C T T C C T C A

420 430 440 450



S V W L R L P S G N S S T SA G T C T G G C T G A G G C T A C C C T C C G G C A A C A G T T C A A C G T C

460 470 480 490

S A H S L S R S S T L L SC A G C C C A T T C T T T G A G C A G A T C C A G C A C T T T A C T T T C T

490 500 510 520

S V R E L S T K F T S R D ST G T G A G G G A A C T T T C C A C A A A G T T C A C C A G C C G G G A T T C

530 540 550 560

S S I F S P L N S * R S S SC G T C G A T T T T T T C T C C C T T G A A T T C C T G A C G A A G C T C G T

570 580 590 600

S Q R S L T S A S V G * IT C A C A G C G T T C C T T A A C T T C T G C T T C G G T G G G C T G A A T T

610 620 630 640

I F S S L A M A P I I R S VT T T T T C T T C T T T G G C A A T G G C T C C T A T C A T C A G G T C G G T

650 660 670



V L R L A I A S G R E L S LT T T T A A G G T T G G C G A T C G C C T C T G G G C G G G A A T T G T C C T

680 690 700 710

L R V W S I S S P * R N ST T G A G G G T T T G G A G C A T A T C A T C C C C C T G A C G G A A C A G T

720 730 740 750

S W E T S S P M R S S S M AT T G G G A A A C A T C T A G T C C C A T G C G T T C T A G T T C C A T G G C G

760 770 780 790

A V W A T R R S H S C L I MG G T T T G G G C C A C T A G G C G A T C G C A C T C C T G T T T G A T C A T G

800 810 820 830

M V S G K S P V N S S P N FG G T T T C C G G T A A A T C C C C G G T G AA C A G T T C C C C C A A T T T T

840 850 860 870

F F L M A A L M L S S V R CT T T C T T A A T G G C A G C C T T G AT G T T G T C A T C G G T G C G C T G T

880 890 900 910



C L A C S W N C F S R S A LT T T GG C C T G T T C C T GG AA T T G T T T C T CC A G A T CC G CC T T G

920 930 940 950

L S S A M V S T R S H L R R SG A G C T C T G CC A TG G T C T C A A C T C G C T C A C A T C TT C G GC GA A

950 960 970 980 990

S H R P I Q Q F P D L I SA G T C AT C G T CC A A T T C A G C A G T T CC C GG A T TT A A TTT C T

990 1000 1010 1020

S L S L I S T H I F P H STTT G A GC T T AA T TT C A A C A C A C AT C T T C C C G C A C A G C

1030 1040 1050 1060

S F Q G * S S G K * R I SC T T CC AA GG AT AG T C C TC C G G A A A GT AA C GG A T C A G T

1070 1080 1090 1100

S L V S S R I R Q Y R H IT TT GG T TT CA T C A C G C A TT C G A CA A TA CC G T C AC A T

1100 1110 1120 1130



I Q H K T T S - G - S NTA C A G C AC A A A A C G A C G TC T Y W A G G T C M C TC A A

1140 1150 1160

N Q T - F - I A S - LA A T C A G A C C T CM TT C G S G A TC G C C T C T TC R C T

1170 1180 1190

L T S S - L K R A Y PT G A C A T C T T C TG C M C TG A AG CG G GC G T A T C

1200 1210 1220

P M P N S R P E -C C A A T G CC G AA T T CC A G G C C C GA G M G

1230 1240 1250

- L - A - - P VG T C T C M C G G CA A G R WG A C C GG T

1260 1270

V F - - - L EHT T T T T YC K G T Y K Y C C T TG AG C AC

1280 1290



HC


A.3 TF in pENTR11 amplified with SP6 sequenc-

ing primers

144

TF-pENTR11.pENTR11-rev.ab1 - Avg. Quality: 0.0A:21%, C:27%, G:21%, T:29%, N:16, Total: 1224

- - - M A I M I L F * LC K S S C C KG G AT G G C AA T AA T G AT T T T A T T T T G A C

10 20 30

L I V T C S L Q Q I D K QC T G AT A G T G AC C T G T T C G T T G C A A CA A A T T G A T A A G C A A

40 50 60 70

Q C F L I M P T L Y K K A GA T G C T T T C T T A T A A T G C C A A C T T T G T A CA A G A A A G C T G G

80 90 100

G S R Y L E L L F V G G C FG G T C T A G A T A T C T C G A G T T A C T C T T C G T C G G A G G T T G C T

110 120 130 140

F H I H D G F F L C I V VT T C C A C A T C C A C G A T G G C T T C T T C C T C T G C A T C G T C G T C

150 160 170 180

V G S I F L S L A E A T L RC G G G A G T A T C T T C C T C A G T C T G G C T G A G G C T A C C C T C C

190 200 210 220



R Q Q F N V S P F F E Q I QC G G C A A C A G T T C A A C G T C A G C C C A T T C T T T G A G C A G A T C C

230 240 250 260

Q H F T F C E G T F H K VC A G C A C T T T A C T T T C T G T G A G G G A A C T T T C C A C A A A G T T

270 280 290

V H Q P G F V D F F S L E FT C A C C A G C C G G G A T T C G T C G A T T T T T T C T C C C T T G A A T

300 310 320 330

F L T K L V T A F L N F CT T C C T G A C G A A G C T C G T C A C A G C G T T C C T T A A C T T C T G C

340 350 360 370

C F G G L N F F F F G N G SC T T C G G T G G G C T G A A T T T T T T C T T C T T T G G C A A T G G C T C

380 390 400 410

S Y H Q V G F K - G D R LC C T A T C A T C A G G T C G G T T T T A A G G K T G G C G A T C G C C T C

420 430 440



L W A G I V L E G L E H I IC T G G G C G G G A A T T G T C C T T G A G G G T T T G G A G C A T A T C A T

450 460 470 480

I P L T E Q L G N I * S HT C C C C C T G A C G G A A C A G T T G G G A A A C A T C T A G T C C C A T

490 500 510 520

H A F * F H G G L G H * A IT G C G T T C T A G T T C C A T G G C G G T T T G G G C C A C T A G G C G A T

530 540 550 560

I A L L F D H G F R * I P GT C G C A C T C C T G T T T G A T C A T G G T T T C C G G T A A A T C C C C G

570 580 590 600

G E Q F P Q F F L N G S LG G T G A A C A G T T C C C C C A A T T T T T T C T T A A T G G C A G C C T T

600 610 620 630

L D V V I G A L F G L F L ET G A T G T T G T C A T C G G T G C G C T G T T T G G C C T G T T C C T G G A

640 650 660 670



E L F L Q I R L E L C H G LA A T T G T T T C T C C A G A T C C G C C T T G A G C T C T G C C A T G G T C

680 690 700 710

L K L A H I F G E V I V Q FC T C A A A C T C G C T C A C A T C T T C G G C G A A G T C A T C G T C C A A T

720 730 740 750

F T Q F P E F N F F E L N FT T C AC G C A G T T C C C G G A A T T T A A T T T C T T T G A G C T T A A T T

760 770 780 790

F K Q H I F P R H S F Q G IT T C A A A C A A C A C A T C T T C C C C C G CC A C A G C T T CC A A GG G AT

800 810 820 830

I V L R K S N R N Q F G F IT A G T CC T C C G G A A A A G T AA CC G G A A T C A G T T T G G T T T C A T C

840 850 860 870

I N R H S D I P S T I P A TC AA CC G C C A T T C C G A C A T A C C G T CC A C A A T A C C A G CCA C A

880 890 900 910



T N D R L L G H S N Q T LA A A C G A CC G T C T TC T A G G T C A C T C A A A T CA G A C C C T T

920 930 940

L H F A I A L S * H L C TT C A C T T C G C G A T C G C T C T C A G C T G A C A T C T C T G C A

950 960 970 980

T E S A * S H A D L QA C T G AA A G C G C GT A G T C A C A T G C A G A T C T C C A G

990 1000 1010

Q R R R L T D S S R FG C G CA G AC G T C T C A C G G A C A G T A G C C G T T T

1020 1030 1040

F C L L T T V T H R -T C T G T C T C C T G A C CA C T G TC A C G C A T C G A M TG

1050 1060 1070

- I D L A G - I F - -G A T C G A T C T C G C T G G C R TG A T T T T T W A G C Y

1080 1090 1100



- R Q F - R N I - HY T C G T C AA T T C S C T C GG A A C A T C C T K C A

1110 1120

H Y - - P Q S D DA C T A T G K A R G G C C T C A G TC A G A T G A

1130 1140 1150

D R F S - Q K CA T A GG T TC A G T C A S C A G A A G TG C

1160 1170

C N G S - Y GC A A T G G G T C T T S T T A C G G C

1180 1190

G * V A * T L *C T A G G T G G C G T A A A C T C T A T G

1200 1210

* R -YG A C G G C C K T AT

1220


A.4 TF in pENTR11 amplified with T7 sequencing

primers

152

TF-pENTR11.pENTR11-fwd.ab1 - Avg. Quality: 0.0A:28%, C:21%, G:27%, T:22%, N:21, Total: 1209

- - L A R A Q I M I L FT M K Y M Y T T A G C T C G G G C C CA A A T AA T G AT T T T A T

10 20 30

F * L I V T C S L Q Q I DT T T T G AC T G A T A G T G A C C T G T T C G T T G CA A C A A A T T G A

40 50 60 70

D K Q C F F I M P T L Y K KA T A A G C A A T G C T T T T T T A T A A T G C CA A C T T T G T A C A A A A

70 80 90 100

K A G F E G D R T L E V LA A A G C A G G C T T C G A A G G A G A T A G A A C C C T G G A A G T T C T G

110 120 130 140

L F Q G P M K V T Q E K L PG T T C C A G G G G C C C A T G A A A G T T A C C C A G G A A A A A T T G C C

150 160 170 180

P D S Q V G L E I E I P AC A G A T A G T C A A G T T G G G C T A G A A A T T G A G A T T C C C G C C

190 200 210 220



A T A S K K V Y E N V V K KC A C A G C G T C G A A A A A A G T T T A T G A G A A C G T C G T C A A A A A

230 240 250

K L T R T V N I P G F R R GA G T T A A C C C G C A C C G T T A A T A T T C C T G G G T T C C G T C G G G

260 270 280 290

G K V P R A I V I Q R L GG G G A A G G T G C C C C G G G C C A T T G T T A T C C A G C G T T T G G G

300 310 320 330

G Q S Y I K A T A I E E L IG A C A A A G C T A T A T C A A G G C C A C G G C C A T T G A A G A G T T G A

340 350 360 370

I D D S I K A A V K Q E EA T C G A C G A C A G C A T T A A A G C C G C G G T T A A G C A A G A A G A A

380 390 400 410

E L P I I G N F S L R S D MA C T A C C C A T C A T T G G C A A T T T T T C C C T G C G C T C G G A C A T

410 420 430 440



M E N L I Q I F D P E A PT G G A A A A C C T A A T T C A A A T C T T T G A C C C T G A A G C G C C C

450 460 470 480

P L T I K V A A D V F P E AC C T C A C C A T T A A G G T G G C A G C G G A T G T G T T C C C C G A A G C

490 500 510 520

A E Y E P E S Y K K I T AC G G A A T A T G A A C C G G A G A G C T A T A A A A A A A T C A C T G C C

530 540 550 560

A Q A E E I E Y S A D A V DC C A G G C G G A A G A A A T C G A A T A C A G T G C C G A T G C C G T T G A

560 570 580 590

D Q W L K G E Q E K R A T LA C C A G T G G C T C A A G G G A G A A C A G G A A A A A C G G G C T A C C C

600 610 620 630

L V P V E D R P A A L G DC T T G T C C C G G T G G A G G A C C G T C C T G C G G C C C T G G G A G A

640 650 660 670



D L A I V D Y A A F Q V A EA T C T G G C C A T T G T G G A C T A C G C C G C T T T C C A G G T G G C A G A

680 690 700 710

E D G Q A G E A I A E V K GA A G A T G G T C A A G C T G G A G A G G C G A T C G C C G A A G T G A A G G

720 730 740 750

G S D F E V T L E D G R F VG G G T C T G AT T T T G A G G T G A C C C T A G A A G A C G G T C G T T T T G

750 760 770 780

V A G I V D G I V G M A VG T G G C T G G T AT T G T G G A C G G T A T T G T C G G AA T GG C G G T T

790 800 810 820

V D E T K L I P V T F P E DT G A T G A A A C C A A A C T G A T T CC G G T T A C T T T T CC GG A G G AC

830 840 850 860

D Y P L E A V A G K M C C LC T A T C C C T TGG A A G C T G T G G C G G G G AA G AT G T G TT G TTT

870 880 890 900



L K L S S R I N S G T A *T G A A A T T A A G C T C A A G A A TT A A T T CC G G A A C T G CC T G

910 920 930 940

* W T M T S R D V S E LG A T G G A C G A T G A C TT C G C G A G AT G T G A G C G A G T T G

940 950 960 970

L R H A E L S D L R T SG A G A C A T G C A G A G C T C A G C G A T C T G A G AA C A T

980 990 1000

S D S N S A R - H I ST C A G A C A G C A A CA G C GC A C G A T G M C A CA T C A G

1010 1020 1030

S C I E N G - V T EG C T G C A T A G A A A A T G G G A C K G T CA C G G A

1040 1050 1060

E Y E H D Q - - I AAA T A C G AA C A T G A T C AA M G A K S S A T C G C T

1070 1080 1090



A M T R M T - H GT A T GA C A C G C A T G A C T G A SC A T GG A

1100 1110

G L - - S S G - CAC T A K T C A C K T C G TC A G G G AK A T

1120 1130

C Q - A F A RT G T C AA C Y A G C A TT C G C G A G

1140 1150

R S L - H - -G A T C A C T A A ASC A C K G A W

1160 1170

- N C - N V *W A G A A C T G C A G R A A TG TAT

1180 1190

* V R A V-TA G G TA C G A GC AG T A T

1200



-T RC


A.5 FFH in pENTR11 amplified with SP6 sequenc-

ing primers

161

pENTR11-FFH.pENTR-rev.ab1 - Avg. Quality: 0.0A:21%, C:26%, G:20%, T:28%, N:51, Total: 1132

- G - I N * F Y F D * * *A K W GGG M AA A TAAA T T G A T T T T A T T T T G A C T G AT A G T

10 20 30

* P V R C N K L I S N A FT G AC C T G T T C GT T G C A A CA A A T T G AT A A G C A A T G C T T T

40 50 60 70

F L * C Q L C T R K L G L DT C T T A T A A T G C CA A C T T T G T A C A A G A A A G C T G G G T C T A G

80 90 100 110

D I S S * S W P K P F F FG A T A T C T C G A G T T A A A G C T G G C C A A A A C C T T T T T T C T T C

120 130 140

F F F F F G L P P P P L * PC T T T T T C T T T T T C G G C T T A C C G C C A C C A C C T C T G T A A C C

150 160 170 180

P R K P G P G * P P N I PC T C G A A A A C C T G G G C C T G G T T G A C C G C C A A A C A T A C C T

190 200 210 220



P P P M P G I A P I P G I PT C C C C C C A T G C C T G G C A T T G C T C C C A T C C C T G G C A T A C C

230 240 250 260

P P G I P P M P I C C I M VC T C C A G G C A T T C C T C C C A T G C C C A T T T G C T G C A T C A T G G

270 280 290 300

V R I L V K L V I S L D TG T C C G C A T C T T G G T G A A A T T G G T G A T T A G C T T A G A C A C A

300 310 320 330

T S V S L * P D P W A M R RA T C G G T T T C A C T A T G A C C C G A T C C T T G G G C T A T G C G A C G

340 350 360 370

R R R L G D L A N K S G FG A C G C C G A C T G G G G G A T T T A G C T A A T A A A T C G G G A T T C

380 390 400 410

F F L S S V V M E L I M A SC T T C C T T T C C T C C G T G G T C A T G G A A C T A A T C A T G G C T T C

420 430 440 450



S V R L S S F S P F S M SC G G T G C G C T T G A G T T C C T T T T C C C C T T T T T C G A T G T C G

460 470 480

S P L L S L F M P G I I F SG C C A C T G C T G A G C T T A T T CA T G C C G G G A A T C A T T T T C A G

490 500 510 520

S R P P R E P I F F I K RG C A G A C C C C C T A G G G A A C C C A T A T T C T T C A T A A A G C G C

530 540 550 560

R I C L M K S S K S K V A SC A T T T G C T T A A T G A A G T C A T C A A A A T C G A A G G T G G C T T C

570 580 590 600

S R I L F C N F S T S P T SC G A G A A T C T T G T T C T G T A A C T T C T C C A C G T C C C C G A C A T

610 620 630

S I A S W A F S T R V S TT C G A T C G C C T C T T G G G C T T T T T C C A C C A G G G T C A G C A C A

640 650 660 670



T S - M F K I R L A R R S GA T C C Y C C A T G T T T A A A A T C C G A C T G GC T A G G C G A T C G G G

680 690 700 710

G W K G C R A S T F S P T PG A T G G A A A G GT T G C A G G G C C T C CA C T T T T T C CC C C A C C C C G

720 730 740 750

P T N L I G W L E I C R - DG A C A A AT T T G A T C G GC T G G C TG G A A A T T T G C C G C M C CG A T

760 770 780 790

D R A A P P L - S P S N L V RT A GG G C T G C A C C G C C C C T A K T A T C C C C AT C T A A C T T G G T AA

800 810 820 830

R I A P - I - - A R G K Y GA G A A T T GC T CCC G K A A T A C M A W T T G C TC G TG GA A A G T A T G

840 850 860 870

G L N - P P L A R M A S T TG G G T T A A A T T G S C C G CCT C T T G C C C G C AT G G CA T C T A C T A

880 890 900 910



T - R D R Q V * - P V - FAC C A G W A G A G A T C GT C A GG T T T AA C M AC C T G TT W A W T T C

920 930 940 950

F - N S A I - - D - L Q T -C G S C AAC T C C GC C AT C W T T G G K G AT M A T CT GC A A A CG AY C

950 960 970 980

- - G - - R P Y R L P - L AC G G S G G A T C R AW T A G A C C GT AT C GA C T C C C A T Y C T TGG

990 1000 1010 1020

A F H - W R A I - R V -G C T T T T C A C C C Y T G GC G GG C TA TT Y C A C G G G T GG S A

1030 1040 1050 1060

- - L - - Q - - - - V - -AT Y M C T G Y C A R G TC AA W C R G C M A Y A W C T G T T G CY M

1070 1080 1090

- I - I G - - - - - - RM A W A T T T Y TA T TG G T C W K G C K S S S S GC S G M W M M C G

1100 1110 1120



R-GTC

1130


A.6 FFH in pENTR11 amplified with T7 sequenc-

ing primers

169

pENTR11-FFH.pENTR-fwd.ab1 - Avg. Quality: 0.0A:26%, C:23%, G:27%, T:22%, N:29, Total: 1045

F - G P Q I M I L F * LT T CM G G G G G C C C CA A A T A A T G AT T T T A T T T T G A C T

10 20 30

L I V T C S L Q Q I D K QT G A T A G T G A C C T G T T C GT T G CA A CA A A T T G AT A A G C A A

40 50 60 70

Q C F F I M P T L Y K K A GA T G C T T T T T T A T A A T G C CA A C T T T G T A CA A A A A A G C A G G

80 90 100

G F E G D R T L E V L F Q GG C T T C G A A G G A G AT A G A A C C C T G G A A G T T C T G T T C C A G G

110 120 130 140

G P M F D A L A D R L E DG G G C C C A T G T T T G A T G C C T T A G C C G A T C G C C T T G A A G A

150 160 170 180

D A W K K L R G Q D K I S EA T G C C T G G A A G A A G C T C C G G G G T C A G G A C A A A A T T A G T G

190 200 210 220



E S N I K E A L Q E V R RG A A A G C A A T A T T A A A G A G G C G C T T C A G G A A G T C A G G A G A

230 240 250 260

R A L L A A D V N L Q V V KA G C C C T G T T G G C G G C G G A C G T G A A T C T C C A G G T G G T C A A

260 270 280 290

K G F I K D V E Q K A L G AA G G G T T T C A T C A A A G A C G T A G A G C A A A A A G C C C T C G G T G

300 310 320 330

A D V I S G V N P G Q Q FG C G G A T G T G A T C A G T G G G G T T A A C C C A G G G C A A C A G T T C

340 350 360 370

F I K I V Y D E L V N L M GC A T C A A A A T T G T T T A C G A C G A A C T G G T A A A C C T G A T G G

380 390 400 410

G E S N V P L A Q A E Q AG G G G A A A G C A A C G T G C C C C T A G C T C A A G C A G A G C A A G C T

420 430 440



A P T V I L M A G L Q G T GT C C C A C C G T C A T T C T T A T G G C C G G C T T G C A G G G G A C C G G

450 460 470 480

G K T T A T A K L A L Y LG G A A A A C C A C C G C C A C G G C C A A A T T A G C C C T C T A T C T C

490 500 510 520

L R K Q K R S A L M V A T DC C G C A A A C A A A A A C G C A G T G C C C T G A T G G T G G C C A C G G A

530 540 550 560

D V Y R P A A I D Q L K TA C G T T T A T C G C C C C G C C G C C A T T G A C C A A C T A A A A A C T

570 580 590

T L G Q Q I D V P V F D L GT C T G G G G C A A C A G A T T G A T G T G C C T G T A T T T G A C C T G G G

600 610 620 630

G S D A N P V E I A R Q G VG C A G T G AT G C C A A C C C G G T G G A A A T T G C C C G C C A A G G G G

640 650 660 670



V E K A K E L G V D T V LG T G G A A A A A G C C A A G G A A T T G G G A G T C G A T AC G G T C T T A

680 690 700 710

L I D T A G - L - I D P Q MA A T C G A TA C C G C C G G T Y G T TT GC A K A T T G A T C C C C A A A T G

720 730 740 750

M M A E L A - I K Q V V K PG AT G GC G G A G T T G G C C G A W A T T A A A C A G G T T GT T A A A C C T

760 770 780 790

P D D T L L V V D A M - G QT G A C G A TA C T C T AC T G G T AG TA G A T GC C A T G A YG G G GC A A

800 810 820 830

Q E A - N L T H T - H E Q LA G A G GC G GY C A A T T T AA C C C AT A C T TT Y C A C G A G C A A TT G

830 840 850 860

L - L P E Q F L P S * M G - LGG K A T TA CC G GA G C A A T T C T T A C C AA G TT A G A T G G G G W T AC

870 880 890 900



L G A V - - Y - - - - F R -C T A G G G G CG G T G C A SY C C T A T CK GW TGC W G M T A T T T C GG G

910 920 930 940

- A D Q F V G W G K K - GG C W G C C G A TC A A T TT G T C G G G T G G G G G A A A A A G K G A G G

950 960 970 980

G P A P - L P - - - - R -G CCC T G C A CC T T Y CC T C C C G AY C G C Y W G C A G Y C GA W T

990 1000 1010 1020

- * - G - - - PFT Y T A A CW T G G G G W Y G G YT Y A C C C T

1030 1040


A.7 TF deletion in pGEM-5 amplified with SP6

primers

176

D12_simontuohy.8.TF-chl-rev.SP6_045.ab1 - Avg. Quality: 0.0A:27%, C:22%, G:22%, T:27%, N:26, Total: 1255

- I G R R K - G K ICM T A T C G G T C G A C G G A A A G K A G G G A A A AT

10 20

I K K K A D R D Q A G V QT T A A G A A A A A G GC G G A C A G G G AT C A A G C C G G C G T C CA A

30 40 50 60

Q G V G K K N F S F T L L *A G G A GT G G G G A A A A A A A A C T T T T C C T T T A C T T T G C T T T A

70 80 90 100

* V N S T L L P T W S H L TA G G T T A A C T C A A C T T T G T T G C C G A C A T G G T C T C A T C T T A

110 120 130 140

T * I Y L S L G G L T P *A C C T A G A T T T A C T T G A G C C T T G G G G G A C T A A C G C C C T A

150 160 170

* D R E G R R L I C P V NA G G A C A G G G A G G G C A G G A G G T T A A T C T G C C C T G T C A A T

180 190 200 210



N L P I P E I S P L V C G GT T T A C C T A T T C C G G A G A T T T C T C C T C T A G T T T G C G G A G G

220 230 240 250

G P V C L * H P K G N L G QG C C C A G T A T G C C T C T G A C A C C C C A A G G G T A A T T T G G G C C

260 270 280 290

Q R V T L E F I R L L S LC A A C G G G T T A C T C T A G A A T T C A T C C G C T T A T T A T C A C T T

300 310 320

L I * A * Q P G V * G H Q *T A T T T A G G C G T A G C A A C C A G G C G T T T A A G G G C A C C A A T

330 340 350 360

* L P * K N Y A P P C H ST A A C T G C C T T A A A A A A A T T A C G C C C C G C C C T G C C A C T C A

370 380 390 400

S S Q Y C C N S L S I L P TA T C G C A G T A C T G T T G T A A T T C A T T A A G C A T T C T G C C G A C

410 420 430 440



T W K P S Q T A * * T * IC A T G G A A G C C A T C A C A A A C G G C A T G A T G A A C C T G A A T C

450 460 470

I A S G I S T L S P C V * YC G C C A G C G G C A T C A G C A C C T T G T C G C C T T G C G T A T A A T A

480 490 500 510

Y L P M V K T G A K K L S IA T T T G C C C A T G G T G A A A A C G G G G G C G A A G A A G T T G T C C A

520 530 540 550

I L A T F K S K L V K L TA T A T T G G C C A C G T T T A A A T C A A A A C T G G T G A A A C T C A C

560 570 580 590

T Q G L A E T K N I F S I NC C C A G G G A T T G G C T G A G A C G A A A A A C A T A T T C T C A A T A A

600 610 620 630

N P L G K * A R F S P * HA A C C C T T T A G G G A A A T A G G C C A G G T T T T C A C C G T A A C A C

630 640 650 660



H A T S C E Y M C R N C R KC G C C A C A T C T T G C G A A T A C A T G T G T A G A A A C T G C C G G A A

670 680 690 700

K S S W Y S L Q S D E N V SA A T C G T C G T G G T A T T C A C T C C A G A G C G A T G A A A A C G T T T

710 720 730 740

S V C S W K T V * Q G * T LT C A G T T T G C T C A T G G A A A A C G G T G T A A C A A GG G T G A A C A C

750 760 770 780

L S H I T S S P S F I A I RC T A T C C C A T A T C A C C A G C T C A C C G T C T T T C A T T G C C A T A C

790 800 810 820

R N S G * A F I T R A R MC G G A AT TC C G G A T G A G C AT T C A T C A C G C G G GC AA G AA T G

830 840 850 860

M * I K A G * T C A Y F S LG T G A A T A A A G G C C G G A T A A A C TT G T G C T T A T T T T T C T T T

860 870 880 890



L R S L K G R N I Q L N G LT A C G GT C T T T A A A A GG C CG T AA T A T C C A G C T G A A C G G T C

900 910 920 930

L V I G T L S T D * M P QC T G G T T A T A G G T A C A T T G A G C A C T G A C T G A A T G C C T C A

940 950 960 970

Q M F F T M P - G Y I T VA A A T G T TC T T T A C G A T G C C A T K GGG AT AT A T C A C G G T G

980 990 1000 1010

V V Y P - I F - H L A S LG G T A T A T CC A G K G A T T T T T T C Y C A T T T A G C T T C C T T A

1010 1020 1030 1040

L A L K - - Y S K - - R *A G C T C T G A A A TY T Y G AT A C T C A A A A A TW C S C C GG T

1050 1060 1070 1080

* - - - H Y - E V E LT A G K G AT Y T W T T C A T T A T G G K G A A G TT G AA C TC

1080 1090 1100 1110



L Y V R S R L I F - K CC T A CG TG C G A T C A C G T C TC A T T T T C S YC A A G T

1120 1130 1140

C R L - S T - QT G C A G G C T T C G W T C A AC G G A WC A G

1150 1160

Q I - Y - - - F R QG A T T W T T T A T Y G S A G K G A Y T T T C G T C

1170 1180 1190

Q I P A C A EC A G A TT CC GG C A T G C G C GG A

1200 1210

E L - L S N C LA A C T G C M G C T G T C C AA T TG C C T

1210 1220 1230

L Y G - L - K -T A T AT G GT G R T C T AT T M A A GT

1240 1250



--T A YG


A.8 TF deletion in pGEM-5 amplified with T7 primers

185

C12_simontuohy.8.TF-chl-fwd.T7_046.ab1 - Avg. Quality: 0.0A:26%, C:21%, G:24%, T:28%, N:17, Total: 1230

R - P A G I S V T E DA G G GGK C C GG C GG G G AT A T C T GT G A C G G A A G AT

10 20 30

D H F A E * I - P G V P VT C A C T T C G C A G A A T A A A T A A W T C C T G G T G T C C C T G T T

40 50 60

V D T G K P W A N F W R K *T G A T A C C G G G A A G C C C T G G G C C A A C T T T T G G C G A A A A T

70 80 90 100

* D V D R H V R G S N F HT G A G A C G T T G A T C G G C A C G T A A G A G G T T C C A A C T T T C A C

110 120 130 140

H H N E I R S L P G V F F EC C A T A A T G A A A T A A G A T C A C T A C C G G G C G T A T T T T T T G

150 160 170 180

E L S R F S G A K E A K MG A G T T A T C G A G A T T T T C A G G A G C T A A G G A A G C T A A A A T G

180 190 200 210



M E K K I T G Y T T V D I SG G A G A A A A A A A T C A C T G G A T A T A C C A C C G T T G A T A T A T C

220 230 240 250

S Q W H R K E H F E A F QC C C A A T G G C A T C G T A A A G A A C A T T T T G A G G C A T T T C A G

260 270 280 290

Q S V A Q C T Y N Q T V Q LG T C A G T T G C T C A A T G T A C C T A T A A C C A G A C C G T T C A G C T

300 310 320 330

L D I T A F L K T V K K N KT G G A T A T T A C G G C C T T T T T A A A G A C C G T A A A G A A A A A T A

340 350 360

K H K F Y P A F I H I L AA A G C A C A A G T T T T A T C C G G C C T T T A T T C A C A T T C T T G C

370 380 390 400

A R L M N A H P E F R M A MC C C G C C T G A T G A A T G C T C A T C C G G A A T T C C G T A T G G C A A

410 420 430 440



M K D G E L V I W D S V HA T G A A A G A C G G T G A G C T G G T G A T A T G G G A T A G T G T T C A

450 460 470 480

H P C Y T V F H E Q T E T FA C C C T T G T T A C A C C G T T T T C C A T G A G C A A A C T G A A A C G T

490 500 510

F S S L W S E Y H D D F RT T T T C A T C G C T C T G G A G T G A A T A C C A C G A C G A T T T C C G

520 530 540 550

R Q F L H M Y S Q D V A C YG G C A G T T T C T A C A C A T G T A T T C G C A A G A T G T G G C G T G T T

560 570 580 590

Y G E N L A Y F P K G F IT A C G G T G A A A A C C T G G C C T A T T T C C C T A A A G G G T T T A T T

600 610 620 630

I E N M F F V S A N P W V ST G A G A A T A T G T T T T T C G T C T C A G C C A A T C C C T G G G T G A G

640 650 660 670



S F T S F D L N V A N M D NG T T T C A C C A G T T T T G A T T T A A A C G T G G C C A A T A T G G A CA

670 680 690 700

N F F A P V F T M G K Y YA A C T T C T T C G C C C C C G T T T T C A C C A T G G G C A A A T A T T A T

710 720 730 740

Y T Q G D K V L M P L A I QT A C G C A A G G C G A C A A G G T G C T G A T GC C G C T G G C G A T T C A

750 760 770 780

Q F I M P F V M A S M S A EA G TT C A T C A T G C C G T T T G T G A T G G CT T C C A T G T C G G C A G

790 800 810 820

E C L M N Y N S T A M S - RG AA T G C T T A A T G A A TT A C AA C A G T A C T G C G A T G A G T GG S A

830 840 850 860

R A G - N F F K A V I G A LAG G G C G G G G C G W AA T T T T T T T A A GGCA G T T A T T GG T G CCC

870 880 890 900



L K R L V A T P N K * * *C TT A A A C G C CT GG T T GC T AC G C C T A A T A A G T G A T A A T A G

900 910 920 930

* R M N S R V P V G Q L PG C G G A T GAA T T CT AG A G T A C C C G TT G G C C A A TT A C C C

940 950 960 970

P L G C Q R H T G P P Q L EC T T G G G G T G TC A G A G G CA T A C T G G G C C T C CG C AA C T A G AG

980 990 1000 1010

E G E S S D S K L T G R LG G GA G A A T CCT CC G A T A G T A AA TT G AC A GG CA G A TT A

1020 1030 1040

L L C L L S - V S P SA C T C T G C C T C C TG T C TA G S G TT AG T C C C A G

1050 1060 1070

S S V I * - M R H - HG CTC A G T A A TC TA G T A R A T GA G A C A T G Y G C A

1080 1090 1100



H K - Y L A V G K FA C A A G T RR T A C TT A G C A G T A GGG A A G T

1110 1120 1130

F S - W S A - CT T T T C C M T C T G G A GC G C T R W C TG

1140 1150

C R I S I S Y SG T C G C AT T T C T A TTT C C T A C T C G

1160 1170

S Q W E S R -G C A A T G G GAG T C A C G T G

1180 1190

- T * I - VG R Y A C T T G A A T C A W G GT A

1200 1210

V L N - L SA C T T A A T S T G C T A T C T

1220



S-T M GT


Appendix B

Antibody creation and testing

B.1 Example trace of SEC run

195

Appendix C

Biophysical characterisation ofSynechocystis sp. PCC 6803 TriggerFactor

C.1 Tables of Crystallisation trial conditions

199

Trigger Factor and its involvement ... - University of Otago

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