Www.company.com GB2.0 GOLDENBRAID 2.0: A comprehensive DNA assembly framework for Plant Synthetic...

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GB2.0

GOLDENBRAID 2.0: A comprehensive DNA assembly framework for Plant Synthetic

Biology and Molecular Farming

Plant Genomics and Biotechnology GroupIBMCP (CSIC-UPV), Valencia

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GB2.0

www.gbcloning.org

Plant Synthetic Biology

Multigenic traits

Plant Genetic Engineering

Single gene traits

MOLECULAR FARMING

The transit from Biotechnology to Synthetic Biology

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GB2.0

The benefits of Modular Design

STA

ND

AR

DIZ

AT

ION

HIGH-THROUGHPUT

FLEXIBILITY

INTERCHANGEABILITY


GB2.0

GB2.0

TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT

1311 12 14 15 16 2101 02

5’ NT TRANSCRIBED REGION 3’ NT

5 UTR LINK 3 UTR

01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)

SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)

CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)

NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)

NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)

OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)

PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)

TL1 TL2 TL4TL3

16 (CT)

17

01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)

16B (3’FS) 17-21 (TER)

GTAG TCTC

amiRNA

01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)

01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)

hpRNA

tasiRNA

TGAC

03

02 (OP)

OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP)

02468

1012141618

PNos 0 μM 5 μM 10 μM P35s

No

rma

lize

d F

Lu

c/R

Lu

c

Grammar

Database

Functionally-tested genetic modules

The GoldenBraid 2.0 Framework

Software tools

Interchangeability

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GB2.0

www.gbcloning.org

Type IIS enzymeType II enzyme

nnnn cctgag ctcgag

Type IIS restriction enzymes

GoldenGate:

Engler, C., R. Gruetzner, R. Kandzia, and S. Marillonnet, PLoS One, 2009. 4(5): p. e5553.Engler, C., R. Kandzia, and S. Marillonnet, PLoS One, 2008. 3(11): p. e3647.

GoldenBraid 1.0:

Sarrion-Perdigones, A., E.E. Falconi, S.I. Zandalinas, P. Juarez, A. Fernandez-Del-Carmen, A. Granell, and D. Orzaez,. PLoS One, 2011. 6(7): p. e21622

Type IIS enzymeType II enzyme

The chemistry behind GoldenBraid

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GB2.0

www.gbcloning.org

The chemistry behind GoldenBraid

Suffix

AATG

Prefix

GCTT

PROMOTER + UTR +ATG CDS TERMINATOR

GGAG AATG GCTT CGCT

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GB2.0

www.gbcloning.org

d t

u

nes

ra n

d

GB 2.0: analogy with the grammar


GB2.0

understand

do

not

ityou

!



GB2.0

you do not understand it!

If you can’t compute it


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GB2.0

www.gbcloning.org

GBparts Transcriptional Units

Genetic Modules

Multipartite

Binary,Iterative,

GB works as a double iterative loop that allows the stepwise assembly of new TUs indefinitely.

GB supports multipartite assemblies of several (up to 8) small DNAparts in a single reaction to create a transcriptional units

The GB 2.0 assembly strategy

Basic Genetic elements

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GB2.0

www.gbcloning.org

pDGB α213 2

pDGB Ω23 21

pDGB α121 LacZ

pDGB Ω11 323

LacZ

LacZ

LacZ

α L

EV

EL

(B

saI r

eact

ion

s) Ω L

EV

EL

(Bsm

BI reactio

ns)

The 4 pDGBs give a double-loop structure (braid)

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GB2.0

www.gbcloning.org

GB database

GB TU2

SEQUENCE

GB partGB part

GB TU1

GB Module

GB Domesticator

GB TU Assembler

GB Binary Assembler

GB part GB part

GB 2.0 software tools


GB2.0

35S Jchain TNos

35S SP TNos

35S SC TNos

CLVL

35S SP TNosCHVH

SP

SP

GoldenBraid 2.0: building a secretory IgA


GB2.0The GB2.0 workflow

35S

SC

TNos

SP

TNos

35S

SP

hSC DOMESTICATION

AGCC

GCTT

AATG

CGCT

GGAG

AATG

GCTTAGCC MULTIPARTITE ASSEMBLY

35S SC TNosSP

PARTS and SUPERPARTS

TRANSCRIPTIONAL UNIT

BINARYASSEMBLY

35S Jchain TNos

35S SC TNosSP

SP

GENETICMODULE


GB2.0

SUBJECT PREDICATE STOP

VERB ART NOUNNOUN

- English sentence

The Grammar

ADJ

Biotechnology

has a hugepotential .


1311 12 14 15 16 2101 02


5´UTR LINK 3´UTR

01-02-03-11-12 (PROM+UTR+ATG)13

(SP) 14-15-16 (CDS) 17-21 (TER)

TL1 TL2 TL4TL3

17

TGAC

03

35S SC TNosSP

- Transcriptional Unit

ART


GB2.0


1311 12 14 15 16 2101 02


5 UTR LINK 3 UTR








TL1 TL2 TL4TL3

16 (CT)

17


16B (3’FS) 17-21 (TER)

GTAG TCTC

amiRNA



hpRNA

tasiRNA

TGAC

03

02 (OP)



1311 12 14 15 16 2101 02


5 UTR LINK 3 UTR








TL1 TL2 TL4TL3

16 (CT)

17


16B (3’FS) 17-21 (TER)

GTAG TCTC

amiRNA



hpRNA

tasiRNA

TGAC

03

02 (OP)

OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP) SC

AGCC GCTT

Domestication of the Secretory Component


GB2.0


1311 12 14 15 16 2101 02


5 UTR LINK 3 UTR








TL1 TL2 TL4TL3

16 (CT)

17


16B (3’FS) 17-21 (TER)

GTAG TCTC

amiRNA



hpRNA

tasiRNA

TGAC

03

02 (OP)



1311 12 14 15 16 2101 02


5 UTR LINK 3 UTR








TL1 TL2 TL4TL3

16 (CT)

17


16B (3’FS) 17-21 (TER)

GTAG TCTC

amiRNA



hpRNA

tasiRNA

TGAC

03

02 (OP)

OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP) 35S SC TNosSP

Multipartite Assembly of the TU encoding the SC

GB DB GB DB GB DB

DOMESTICATED


GB2.0

pDGB_Ω23 2LacZ

TU SC

1 3

TU JC

3 2

1

3 2

Binary Assembly of the TUs encoding the SC and the JC


GB2.0

pDGB_α1

21 3

TU HC TU LC

31

3 2

1

LacZ

TU JCTU SC

3

Binary Assembly of the preassembled genetic modules


GB2.0

Secretory IgA construction

4 TUs: 13kb


GB2.0

The major benefit of GB2.0 framework: interchangeability

Every story has an end, but with GB every ending is just a new beginning.


GB2.0

GB2.0 is a convenient framework for Plant Synthetic Biology, including Molecular Farming and Metabolic

Engineering

To learn more: www.ibmcp.upv.es/FGBwww.gbcloning.org

Conclusions

Hands-on course on multiple gene assembly technologies for metabolic engineering and fruit biotechnology

Valencia (Spain), September 9th to 13th, 2013

Organized by COST Actions FA1006 (PlantEngine) and FA1106 (QualityFruit)

http://www.plantengine.eu/node/156; http://qualityfruit.inp-toulouse.fr/en/training/dna-assembly.html

http://www.ibmcp.upv.es/FGB

http://www.gbcloning.org/

http://www.plantengine.eu/node/156

http://qualityfruit.inp-toulouse.fr/en/training/dna-assembly.html

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THANK YOU!

IBMCPAlejandro Sarrión

Javier FormentPaloma JuárezEstefanía Huet

Asun FernándezJose Manuel Julve

Diego Orzáez

Fruit genomics and Biotechnology Group.

www.ibmcp.upv.es/FGB

COMAVPeio Ziarsolo

Jose Blanca

http://www.ibmcp.upv.es/

Www.company.com GB2.0 GOLDENBRAID 2.0: A comprehensive DNA assembly framework for Plant Synthetic...

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