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"GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology". Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia. ….in most labs multigene assembly is a bottleneck in the development of new complex plant traits. Combining traits is easy.

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slide1
"GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology"

Fruit genomics and Biotechnology Group

Diego Orzaez, IBMCP, CSIC-UPV, Valencia

slide2
….in mostlabsmultigeneassemblyis a bottleneck in thedevelopment of new complexplanttraits.
slide3
Combiningtraitsiseasy.

Ideally, plantgeneticparts and modules are betterassembled at DNA level

Ideal features of a DNA assemblysystemforPlantBiotechnology:

-Standarized.

-Reusable pieces and modules.

-Simple (rules and toolkit).

-Efficient

slide4
Combiningtraitsiseasy

TheGoldenBraidAssembly

An iterative cloning system for standardized assembly of reusable genetic modules.

slide5
Combiningtraitsiseasy.

GoldenBraid makes use of type IIS restriction enzymes

ctcgag

nnnn cctgag

Type II enzyme

Type IIS enzyme

slide6
Combiningtraitsiseasy.

GoldenBraid is based on Golden Gate, an assembly system described previously:

TheGoldenGateAssemblySystem

GoldenGate

One tube-one reaction

GoldenGateis a extreamlyefficient, potentialyseamlessmultipartiteassemblysystem

Engler C, et al (2009) PLoS One 4: e5553.

Engler C, Kandzia R, Marillonnet S (2008) PLoS One 3: e3647.

slide7
Combiningtraitsiseasy.

Golden Gate functions with TypeIIS restriction enzymes

pENTR_Part1

pENTR_Part2

1

3

3

2

BsaI

PART 1

BsaI

BsaI

PART 2

BsaI

1

BsaI

Lac Z

BsaI

2

pDEST

slide8
Combiningtraitsiseasy.

TheGoldenGateAssemblySystem

One tube-one reaction:

Ligase + RE (cycles 16ºC and 37ºC)

PART 1

PART 2

pDEST

Seamless, multipartite assembly

High efficiency

Combinatorial

slide9
CDS

4

PROM

TERM

Combiningtraitsiseasy.

Golden Gate opened the way to multipartite assembly of standarized DNA pieces

pENTR_Term

4

3

BsaI

BsaI

PROM

pENTR_Term

pENTR_PROM

3

2

BsaI

BsaI

1

BsaI

BsaI

ctta

atgg

tgaa

ttgg

4

3

1

2

CDS

TERM

2

BsaI

Lac Z

BsaI

1

pDEST

RB

LB

slide10
Combiningtraitsiseasy.

COLLECTION OF STANDARIZED PARTS

3-2

1-4

4-3

PROM

ctta

atgg

tgaa

ttgg

4

3

1

2

CDS

TERM

RB

LB

pEXPRESSION

slide11
Combiningtraitsiseasy.

END OF THE STORY…

Golden Gate did not allow multigene assembly.

PROM

ctta

atgg

tgaa

ttgg

4

3

1

2

CDS

TERM

RB

LB

pEXPRESSION

slide12
Combiningtraitsiseasy.

ACTUALLY, IT WAS THE START OF THE STORY…

TheGoldenBraidAssemblySystem

Sarrión-Perdigones et al (2011) PloS One 6(7): e21622.

slide13
Combiningtraitsiseasy.

END OF THE STORY…UNLESS

PROM

ctta

atgg

tgaa

ttgg

4

3

1

2

BsbI

A

CDS

TERM

C

BsbI

pEXPRESSION

LB

RB

slide14
Combiningtraitsiseasy.

END OF THE STORY…UNLESS

Adding a second type IIS flanking sites to the destination plasmid, we can release the entire transcription unit (TU) and assemble it with another TU .

PROM

TRANSCRIPTIONAL UNIT 1

ctta

atgg

tgaa

ttgg

4

3

1

2

BsbI

A

CDS

TERM

C

BsbI

LB

RB

pEXPRESSION

slide15
GoldenBraid assembly

Combiningtraitsiseasy.

pENTR_Part1

pENTR_Part1

TU 1

TU 2

C

B

BsbI

A

C

BsbI

BsbI

BsbI

A

BsbI

Lac Z

BsbI

B

pDEST

End of the story again…?

This strategy allows joining two Transcriptional Units, but again we run out of restriction sites, what brings us to a dead end.

slide16
Combiningtraitsiseasy.

GoldenBraid uses a simple toolbox of 4 destination plasmids

(pDGBs)

A

1

Z

2

C

1

A

Z

B

3

pDGB_A12C

pDGB_1AB3

To solve this dead end we developed GoldenBraid, an iterative system that allows indefinite assembly of new TUs.

Level αplasmids

Level Ωplasmids

3

A

Z

B

2

C

1

Z

2

B

pDGB_1AB3

pDGB_C12B

slide17
Combiningtraitsiseasy.

TheGoldenBraidAssemblySystem

TU1

P1 P2 P3

Golden GATE

TU1

TU2

GoldenBraid

P1 P2 P3

TU2

Golden GATE

16h to colonies!!

GoldenBraidtakesmultipartiteGoldengatecompositeparts and assemblythembinarely

slide18
GoldenBraid assembly

Combiningtraitsiseasy.

slide19
Combiningtraitsiseasy.

The 4 pEDGBs give a doble-loop structure (braid)

A

1

Z

2

C

1

A

Z

B

3

pDGB_A12C

pDGB_1AB3

3

A

Z

B

2

C

1

Z

2

B

pDGB_1AB3

pDGB_C12B

slide20
Combiningtraitsiseasy.

Each destination plasmid performs a different “braid twist”

1

A

Z

B

3

pDGB_1AB3

TU1

3

1

C

A

TU2

B

C

slide21
Combiningtraitsiseasy.

In each “Braid twist” 2 composite parts are assembled together

A

1

Z

2

C

pDGB_A12C

A

C

TU1

TU2

3

1

TU3

3

2

slide22
Combiningtraitsiseasy.

GoldenBraid assemblies are “reusable” and “ready to transform”

TU1

TU2

TU3

TU4 TU5

3

A

Z

B

2

pDGB_1AB3

slide23
1

A

Z

B

3

pDGB_1AB3

Combiningtraitsiseasy: a real example

Silencing

Supressor

Blue Fluorescent

Protein

Green Fluorescent

Protein

Red Fluorescent

Protein

R

B

G

P

A

C

A

C

C

B

C

B

35S-RED-NT

35S-BFP-NT

35S-GFP-NT

35S-P19-NT

G

B

1

3

G

A

C

B

C

B

slide24
Combiningtraitsiseasy: a real example

Silencing

Supressor

Blue Fluorescent

Protein

Green Fluorescent

Protein

Red Fluorescent

Protein

R

B

G

P

A

C

A

C

C

B

C

B

35S-RED-NT

35S-BFP-NT

35S-GFP-NT

35S-P19-NT

Only one day work up to 2X TU assembly!!

G

B

1

3

R

A

C

3

R

P

2

P

C

B

3

A

Z

B

2

pDGB_1AB3

slide25
Combiningtraitsiseasy: a real example

Silencing

Supressor

Blue Fluorescent

Protein

Green Fluorescent

Protein

Red Fluorescent

Protein

R

B

G

P

A

C

A

C

C

B

C

B

35S-RED-NT

35S-BFP-NT

35S-GFP-NT

35S-P19-NT

A

1

Z

2

C

pDGB_A12C

G

B

1

3

A

C

G

B

R

P

3

R

P

2

slide26
Combiningtraitsiseasy: a real example

GB assembly ensures coordinated expression in N. benthamiana

G

B

R

P

A

C

B

P

R

+

R

C

B

C

B

+

A

C

A

C

+

slide27
Combiningtraitsiseasy: a real example

GB assembly ensures coordinated expression in N. benthamiana

G

B

R

P

A

C

B

P

R

+

R

C

B

C

B

+

A

C

A

C

+

slide28
Combiningtraitsiseasy.

GoldenBraid allows indefinite binary assemblies of reciclable genetic composites made of standard genetic parts: a versatile tool for complex genetic engineering and synthetic biology in plants.

slide29
Fruit genomics and Biotechnology Group.

Alejandro Sarrión-Perdigones

Paloma Juárez

JoseManuel Julve

Asun Fernández

Silvia Presa

Antonio Granell

Diego Orzáez

¡GRACIAS!

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