"GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and...

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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

Transcript of "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and...

Page 1: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

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

Fruit genomics and Biotechnology GroupDiego Orzaez, IBMCP, CSIC-UPV, Valencia

Page 2: "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.

Page 3: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Ideal features of a DNA assembly system for Plant Biotechnology:

-Standarized.

-Reusable pieces and modules.

-Simple (rules and toolkit).

-Efficient

Combining traits is easy.

Ideally, plant genetic parts and modules are better assembled at DNA level

Page 4: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Combining traits is easy

The GoldenBraid Assembly

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

Page 5: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Type IIS enzymeType II enzyme

nnnn cctgag ctcgag

GoldenBraid makes use of type IIS restriction enzymes

Combining traits is easy.

Page 6: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

The Golden Gate Assembly System

GOLDEN GATE

Golden Gate is a extreamly efficient, potentialy seamless multipartite assembly system

One tube-one reaction

Combining traits is easy.

Engler C, et al (2009) PLoS One 4: e5553.Engler C, Kandzia R, Marillonnet S (2008) PLoS One 3: e3647.

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

Page 7: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PART 11 PART 2

Lac Z BsaIBsaI

BsaIBsaI BsaIBsaI 33

1

2

2

pENTR_Part1 pENTR_Part2

pDEST

Combining traits is easy.

Golden Gate functions with TypeIIS restriction enzymes

Page 8: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PART 1PART 2

pDEST

Seamless, multipartite assemblyHigh efficiencyCombinatorial

One tube-one reaction:Ligase + RE (cycles 16ºC and 37ºC)

Combining traits is easy.

The Golden Gate Assembly System

Page 9: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

BsaI BsaI

1 2

pENTR_PROM

pDEST

CDS4BsaI BsaI

pENTR_Term

PROM 4

3

TERM3BsaI BsaI

pENTR_Term

21

PROM CDS TERM1 24 3atgg tgaactta ttgg

LB RB

BsaI BsaILac Z

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

Combining traits is easy.

Page 10: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PROM CDS TERM1 2

pEXPRESSION

COLLECTION OF STANDARIZED PARTS

1-4

4-3

3-2

4 3atgg tgaactta ttgg

LB RB

Combining traits is easy.

Page 11: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PROM CDS TERM1 2

pEXPRESSION

END OF THE STORY…

4 3atgg tgaactta ttgg

LB RB

Combining traits is easy.

Golden Gate did not allow multigene assembly.

Page 12: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

ACTUALLY, IT WAS THE START OF THE STORY…

Combining traits is easy.

The GoldenBraid Assembly System

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

Page 13: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PROM CDS TERM1 2

pEXPRESSION

4 3atgg tgaactta ttgg

BsbI A BsbIC

END OF THE STORY…UNLESS

LB RB

Combining traits is easy.

Page 14: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

PROM CDS TERM1 2

pEXPRESSION

4 3atgg tgaactta ttgg

BsbI A BsbIC

TRANSCRIPTIONAL UNIT 1

LB RB

Combining traits is easy.

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 .

Page 15: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Lac Z BsbIBsbI

BsbI BsbI

GoldenBraid assembly

pENTR_Part1

pDEST

TU 1A C BsbI BsbI

pENTR_Part1

TU 2C B

A B

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.

Combining traits is easy.

Page 16: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_C12B1 2C BZ

pDGB_1AB3

pDGB_1AB33 2A BZ

pDGB_A12C

Level α plasmids Level Ω plasmids

1 3A BZ1 2A CZ

GoldenBraid uses a simple toolbox of 4 destination plasmids(pDGBs)

Combining traits is easy.

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

Page 17: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

GOLDEN GATE

GoldenBraid takes multipartite Golden gate composite parts and assembly them binarely

16h to colonies!!

GOLDEN GATE

P1 P2 P3 TU1

TU2P1 P2 P3GoldenBraid

TU1 TU2

The GoldenBraid Assembly System

Combining traits is easy.

Page 18: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

GoldenBraid assemblyCombining traits is easy.

Page 19: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_C12B1 2C BZ

pDGB_1AB31 3A BZ

pDGB_1AB33 2A BZ

pDGB_A12C1 2A CZ

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

Combining traits is easy.

Page 20: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Each destination plasmid performs a different “braid twist”

TU1

TU2

pDGB_1AB31 3A BZ

Combining traits is easy.

A C

C B

31

Page 21: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_A12C1 2A CZ

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

TU3

TU1 TU2

Combining traits is easy.

31

3 2

CA

Page 22: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

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

TU3TU1 TU2

TU4 TU5

pDGB_1AB33 2A BZ

Combining traits is easy.

Page 23: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_1AB31 3A BZ

GA C

BC B

1 3G B

RA C

35S-RED-NT

Red Fluorescent Protein

B

35S-BFP-NT

Blue Fluorescent Protein

C BP

35S-P19-NT

SilencingSupressor

C BGA C

35S-GFP-NT

Green Fluorescent Protein

Combining traits is easy: a real example

Page 24: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_1AB33 2A BZ

1 3G BRA C

PC B3 2R P

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

RA C

35S-RED-NT

Red Fluorescent Protein

B

35S-BFP-NT

Blue Fluorescent Protein

C BP

35S-P19-NT

SilencingSupressor

C BGA C

35S-GFP-NT

Green Fluorescent Protein

Combining traits is easy: a real example

Page 25: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

pDGB_A12C1 2A CZ

1 3G BRA C

3 2R P

G B P

RA C

35S-RED-NT

Red Fluorescent Protein

B

35S-BFP-NT

Blue Fluorescent Protein

C BP

35S-P19-NT

SilencingSupressor

C BGA C

35S-GFP-NT

Green Fluorescent Protein

Combining traits is easy: a real example

Page 26: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

RA CG B P

RA C BC B RA C PC B+ + +

GB assembly ensures coordinated expression in N. benthamiana

Combining traits is easy: a real example

Page 27: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

RA CG B P

RA C BC B RA C PC B+ + +

GB assembly ensures coordinated expression in N. benthamiana

Combining traits is easy: a real example

Page 28: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

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.

Combining traits is easy.

Page 29: "GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology" Fruit genomics and Biotechnology Group Diego Orzaez, IBMCP, CSIC-UPV, Valencia.

Alejandro Sarrión-PerdigonesPaloma JuárezJose Manuel JulveAsun FernándezSilvia PresaAntonio GranellDiego Orzáez

¡GRACIAS!

Fruit genomics and Biotechnology Group.