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"GoldenBraid: A Standarized DNA Assembly System for Plant Synthetic Biology"
Fruit genomics and Biotechnology GroupDiego Orzaez, IBMCP, CSIC-UPV, Valencia
….in most labs multigene assembly is a bottleneck in the development of new complex plant traits.
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
Combining traits is easy
The GoldenBraid Assembly
An iterative cloning system for standardized assembly of reusable genetic modules.
Type IIS enzymeType II enzyme
nnnn cctgag ctcgag
GoldenBraid makes use of type IIS restriction enzymes
Combining traits is easy.
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:
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
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
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.
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.
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.
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.
PROM CDS TERM1 2
pEXPRESSION
4 3atgg tgaactta ttgg
BsbI A BsbIC
END OF THE STORY…UNLESS
LB RB
Combining traits is easy.
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 .
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.
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.
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.
GoldenBraid assemblyCombining traits is easy.
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.
Each destination plasmid performs a different “braid twist”
TU1
TU2
pDGB_1AB31 3A BZ
Combining traits is easy.
A C
C B
31
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
GoldenBraid assemblies are “reusable” and “ready to transform”
TU3TU1 TU2
TU4 TU5
pDGB_1AB33 2A BZ
Combining traits is easy.
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
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
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
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
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
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.
Alejandro Sarrión-PerdigonesPaloma JuárezJose Manuel JulveAsun FernándezSilvia PresaAntonio GranellDiego Orzáez
¡GRACIAS!
Fruit genomics and Biotechnology Group.