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

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

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

  2. ….in mostlabsmultigeneassemblyis a bottleneck in thedevelopment of new complexplanttraits.

  3. 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

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

  5. Combiningtraitsiseasy. GoldenBraid makes use of type IIS restriction enzymes ctcgag nnnn cctgag Type II enzyme Type IIS enzyme

  6. 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.

  7. 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

  8. Combiningtraitsiseasy. TheGoldenGateAssemblySystem One tube-one reaction: Ligase + RE (cycles 16ºC and 37ºC) PART 1 PART 2 pDEST Seamless, multipartite assembly High efficiency Combinatorial

  9. 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

  10. 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

  11. 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

  12. Combiningtraitsiseasy. ACTUALLY, IT WAS THE START OF THE STORY… TheGoldenBraidAssemblySystem Sarrión-Perdigones et al (2011) PloS One 6(7): e21622.

  13. Combiningtraitsiseasy. END OF THE STORY…UNLESS PROM ctta atgg tgaa ttgg 4 3 1 2 BsbI A CDS TERM C BsbI pEXPRESSION LB RB

  14. 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

  15. 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.

  16. 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

  17. Combiningtraitsiseasy. TheGoldenBraidAssemblySystem TU1 P1 P2 P3 Golden GATE TU1 TU2 GoldenBraid P1 P2 P3 TU2 Golden GATE 16h to colonies!! GoldenBraidtakesmultipartiteGoldengatecompositeparts and assemblythembinarely

  18. GoldenBraid assembly Combiningtraitsiseasy.

  19. 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

  20. Combiningtraitsiseasy. Each destination plasmid performs a different “braid twist” 1 A Z B 3 pDGB_1AB3 TU1 3 1 C A TU2 B C

  21. 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

  22. Combiningtraitsiseasy. GoldenBraid assemblies are “reusable” and “ready to transform” TU1 TU2 TU3 TU4 TU5 3 A Z B 2 pDGB_1AB3

  23. 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

  24. 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

  25. 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

  26. 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 +

  27. 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 +

  28. 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.

  29. 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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