A u s t r a l i a ’ s G r o w i n g F u t u r e

The Xylanase Transient Quantification System

Claudia Vickers

Transient vs. Stable Transformation

• Transient transformation– DNA extra-chromosomal– Seen over several days following transformation

• Stable transformation– DNA integrated into chromosome– Demonstrated by transferral of transgene to offspring– Segregation occurs in offspring

Transient Transformation as a Tool

• Quick and easy (days compared to months)

• Not prone to position-of-integration effects

• Cheap and less labour-intensive than producing transgenics (esp. cereals)

• Measure tissue specificity and expression strength

• But – expression patterns not always reflected in stable transgenics

Reporter Genes

• Genes which allow easy detection of expression to confirm occurrence of transformation

• Different reporters useful for different experiments

• Properties of the reporter gene must be tuned to the experiment

Desirable Features

• Short coding sequence (easy to manipulate)

• Low (preferably no) endogenous activity in plants

• No endogenous substrates in plants

• Easy, quantitative assay

• Cell autonomous

• Tolerate terminal fusions (purification)

• Active and stable under a range of cellular conditions

• Cheap!!!

Reporter Genes

GENE PRODUCT DETECTION

PROS CONS

GUS(uidA)

-glucuronidase

Colour reaction, fluorescence

Extremely well characterisedExcellent assay systems availableQualitative, quantitative

Many reports of endogenous GUS-like activityProblems with cell autonomyQuenching

LUC(luc)

Luciferase(firefly/ bacterial)

Luminescence (light emission)

Extremely sensitiveQualitative, quantitative

Very expensiveVery labileCannot increase sensitivity

antRC

Regulatory proteins

Anthocyanin pigment

Cell-autonomousNo substrate requiredNon-destructive

Not for quantificationToxicity problems

GFP(gfp)

Green fluorescent protein

Fluorescence

Cell-autonomousNon-destructiveNo substrate requiredPictures look great!

Problems with quantificationChlorophyll autofluorescence

Transient Analysis

• Tissue specificity– How many tissues??– Limited to tissues that can be transformed– Tissue specificity may require chromosomal integration– Most reporters will suffice

• Quantification– Assess promoter strength– Test effect of introns, enhancers, 5 and 3 UTRs etc.– Not all reporters are suitable

Expression Strength

1. Focus counting– Co-transform with GFP and GUS plasmids– Count foci (GFP and GUS)– Expression strength = ratio of GFP:GUS– Problems with threshold effects

1m gold particles

Plasmids containing construct and reporter

Quick, cheap, easy

Focus Counting

Focus Counting: Threshold Effect

0 bp3009001200 bp 600 0 0.5 1 1.5GFP:GUS

Barley

Wheat

GFPXE BMS

TSS

UTR

D1 D3

Actin

OG

OG D1

OG D3

OG X

OG S

OG M

Barley

Wheat

Focus Counting: Threshold Effect

0

10

20

30

40

50

60

70

80

P1 P2 P3 P4 P5 P6 P7

Fo

cus

Inte

nsi

ty (

RL

U)

Detection threshold

Saturation threshold

Focus Counting – Threshold Effect

1 2 3 4 5 6

Promoter

1 2 3 4 5 6

Promoter

Fo

cus

Inte

nsi

ty

0

20

40

60

80

Nu

mb

er o

f F

oci

Average Focus Insensity

Number of Foci

B

Expression Strength

• Focus counting– Co-transform with GFP and GUS plasmids– Count foci (GFP and GUS)– Expression strength = ratio of GFP:GUS– Problems with threshold effects

• Protein extraction and reporter quantification– Shoot with GUS and LUC plasmids– Extract protein from tissues– Quantitative reporter gene assays– Expression strength = ratio of reporter activity– Problems….

Schledzewski & Mendel 1994

Reporters for Quantitative Assay

• GUS: Fluorometric, time-response assay– Very sensitive (can increase incubation time)– Slope over time – good statistical significance– Problems with endogenous activity and/or quenching

(particularly in leaf extracts)

• LUC: Luminometric assay– Extremely labile– No option to increase sensitivity– Expensive (substrate, co-factors, detection equipment)

Solution:

• Thermostable XYN developed by gene shuffling• Codon optimised• Stable transformants generated• Time-response assays possible (increase sensitivity)• Substrate:AZCL-xylan

– Minor problem: insoluble– Other potential substrates: 4-MU-xylan (fluorometric), soluble

colorimetric substitute

• Cheap Cheap Cheap (like the birdie)!• Used in concert with GUSPlus greatly incr. sensitivity

XylanaseXylanase

Physical Optima

• pH = 4.5

• Temp. = 40C

• Linear response to enzyme concentration

• Substrate saturates at 0.5%

pH Response @ 40 C

0

1

2

3

4

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

pH

Ab

sorb

ance

(59

0 n

m)

Temperature Response @ pH = 4.5

0

1

2

3

4

0 20 40 60 80

Temperature (Degrees C)

Ab

so

rba

nc

e @

59

0 n

m

Enzyme Concentration

R2 = 0.9924

0

1

2

3

4

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Extract Dilution

Ab

sorb

ance

@ 5

90 n

m

Substrate Concentration

0.0

0.5

1.0

1.5

2.0

2.5

0 0.5 1 1.5 2 2.5

AZCL-Xylan (%)

Ab

sorb

ance

@ 5

90 n

m

Time Response

Time Response - P/100

y = 0.0004x + 0.0377

R2 = 0.99090.00

0.20

0.40

0.60

0.80

0 500 1000 1500 2000

Time (min)

Ab

so

rba

nc

e (

A5

70

)

• Linear over time

• Up to 48 hr

• Allows accurate quantification of weak promoters

High-Throughput

• Colour intensity for each well depends on:– Transformation efficiency

– Concentration of sample

– Time of incubation

• Standardised to internal control (GUS)

Deletion Analysis- Focus Counting

Actin

OG

OG X

OG S

OG M

OG D3

OG D1

0 bp3009001200 bp 600 0 0.5 1 1.5GFP:GUS

Barley

Wheat

GFPXE BMS

TSS

UTR

D1 D3

Actin

OG

OG D1

OG D3

OG X

OG S

OG M

Barley

Wheat

Deletion Analysis – GUS:XYN

100 200 300 400 500

GUSPlus :Xylanase100 200 300 400 500

GUSPlus :Xylanase

0 bp3009001200 bp 600 0 bp3009001200 bp 600 0 bp3009001200 bp 600

Globulin

Hordein

Glo X

Glo S

Glo M

Negative

Glo D1

Glo D2

Glo D3

Summary: XYN/GUSPlus

• Quick (transient vs. stable)• User-friendly• Accurate• Cheap• Sensitive

Vickers, C.E.; Xue, G.-P.; Gresshoff, P.M. (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Rep. 22(2):135-140

 

Xylanase Assaysin Plant TissuesClaudia Vickers [claudia.vickers@csiro.au]

Caveat: It is assumed that the readers have read the paper entitled, ”A synthetic xylanase as a novel reporter in plants” (Plant Cell Reports 22(2):135-140, 2003) before reading these notes.

 

IntroductionReporter Genes UsedDetailed MethodSolutionsReferences

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IntroductionQuantification of the effect of promoter regions on reporter gene expression in transient assays can be used to gain information about how a particular section of DNA drives gene expression in isolation from a chromosomal context. There are advantages and disadvantages associated with using transient analysis. The obvious disadvantage is that promoters do not always behave in the same fashion when integrated into plant DNA. Conversely, one can obtain data that is not affected by position-of-integration effects. In addition, the method is quick and simple compared to the production of transgenic plants. As noted previously, this is particularly useful when investigating promoter activity in species for which the transformation efficiency is relatively low.

Accurate quantification of transient expression requires extraction of protein and quantitative analysis of reporter gene activity. Extremely high sensitivity of reporter gene assays is required because of the dilution of the gene product during protein extraction (the vast majority of cells in bombarded tissues are untransformed). This is generally achieved through extreme sensitivity of detection of the reporter gene product or of the product of the reporter gene’s activity. Enzymatic activity is a practical necessity for this kind of detection. In practice, this results in amplification of the signal. Enzymatic systems that allow extension of incubation periods and consequent accumulation of the cleavage product are preferable, as this allows for further increases in assay sensitivity.

http://www.pi.csiro.au/XylanaseAssays/index.htm

Ordering the XYN Assay Kit

PLASMID ORDER FORM

A charge of AUD $100 per package is made solely to cover costs of production and postage. Please return this form, together with the attached materials transfer agreement, to the ARC Centre of Excellence for Integrative Legume Research by fax or post. Please print clearly. Name: Dr/ Mr/ Mrs / Ms _____________________________________________________________________

Institute: ______________________________________________________________________________

Postal Address: ______________________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

Street Address: ______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

Phone: __________________________________

Fax: __________________________________

Email: __________________________________

Plasmids (pUbiSXR, pActXYN and pUbiGUSPlus) are supplied as 100 ng samples blotted on paper. Please note that we cannot send plasmids until a signed copy of the attached materials transfer agreement has been received. An invoice for AUD $100 will be included with the package. Please return signed and dated form to Ian Harris, Chief Operating Officer, Centre for Integrative Legume Research, John Hines Building (62), The University of Queensland, Brisbane QLD AUSTRALIA 4072. Forms may be faxed to +61 7 3365 3556.

ARC Centre of Excellence for Integrative Legume Research

John Hines Building (62) The University of Queensland St Lucia Brisbane, AUSTRALIA 4072 Ph: +61 7 3365 3550 Fax: +61 7 3365 3556 Email: director.cilr@uq.edu.au

MATERIAL TRANSFER AGREEMENT (MTA)

This material transfer agreement (MTA) is made between (name)_____________________________________,

an employee of (institute and street address) ____________________________________________________

__________________________________________________________________________________________

(‘the Recipient’) and The Centre for Integrative Legume Research at the University of Queensland (“the University”). This MTA covers transfer of the following Materials:

Xylanase assay plasmid vector kit:

- pUbiSXR - pActXYN - pUbiGUSPlus

This material is provided with no warranties of any kind, express or implied, and with no representation that use or supply of these materials will not infringe the rights of any third party, including intellectual property ri ghts. To the full extent permitted by the laws of Australia or of any state or territory of Australia having jurisdiction, any conditions or warranties imposed under legislation are hereby excluded. The University and its employees, past or present, cannot be held liable for any use of the material transferred according to this agreement. The Recipient agrees to the following conditions:

1. These plasmid vectors may be used freely for academic and non-profit research. 2. Use shall be duly acknowledged citing this internal non-commercial non-exclusive license from the

University. 3. Commercial Use: A license from the University is required (i) for any commercial use of the plasmid

vectors including research and production purposes regardless of academic or non-profit status and (ii) for use by any commercial entity. Information about commercial licenses for the Plasmid Vectors may be obtained from CILR at The University of Queensland

4. Plasmids may be distributed to third parties, however, the University must be notified prior to transfer and a copy of this MTA must be signed and returned to the University before the plasmids are transferred.

5. Use of the xylanase (XYN) gene must be acknowledged in resulting publications. The citation is: Vickers, C.E.; Xue, G.P.; Gresshoff, P.M. (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Reports 22[2]: 135-140

6. The GUSPlus gene in pUbiGUSPlus is protected by patents belonging to CAMBIA. Terms for the use of this gene can be obtained from CAMBIA (GPO Box 3200, Canberra, ACT 2601, Australia; website http://www.cambia.org/ or email vectors@cambia.org).

7. Other components of these vectors may be similarly protected by other parties. Obtaining licences for these components (if required) is the responsibility of the user.

8. The Recipient agrees to hold harmless the University, and any of its employees (past or present), agents and officers from any and all liabilities or claims resulting from transfer to, use, or storage of the Materials by the Recipient.

9. This Agreement cannot be assigned. 10. This Agreement is subject to Australian Law and exclusive interpretation by the Australian Courts.

I, (name)________________________________, an authorised officer of (institute) ______________________

___________________________________________________________________________________________,

have read and agree to be bound by the above terms on behalf of and for (institute) ________________________

___________________________________________________________________________________________.

Signed: ……………………………………………. Name: ………………………………….……………………

(Please print clearly) Position:………………………………………………………………………………. Date: ……………………

Xylanase Assay Kit

XYLANASE ASSAY PLASMID VECTOR KIT Please find enclosed the following plasmids as requested:

pUbiSXR

pActXYN

pUbiGUSPlus

Plasmids are spotted onto paper in a solution of 50% sucrose in TE buffer with loading dye. Re-suspend the blue spots in 5 –10 l of water or TE by pipetting back and forth several times. Transfer to a tube and dilute one in ten before transforming into E. coli cells. Full plasmid sequences with annotations can be found in GenBank under the following accession numbers:

AY452753 (pUbiSXR)

AY452735 (pActXYN)

AY452736 (pUbiGUSPlus)

Best wishes and good luck with your research!

ARC Centre of Excellence for Integrative Legume Research

John Hines Building (62) The University of Queensland St Lucia Brisbane, AUSTRALIA 4072 Ph: +61 7 3365 3550 Fax: +61 7 3365 3556 Email: director.cilr@uq.edu.au

Thanks

• Supervisors– Gangping Xue (CSIRO)– Peter Gresshoff (UQ)

• Scholarship– Grains Research and Development Corporation