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

2. ….in most labs multigene assembly is a bottleneck in the development of new complex plant traits.

3. 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

4. Combining traits is easy The GoldenBraid Assembly An iterative cloning system for standardized assembly of reusable genetic modules.

5. Type IIS enzymeType II enzyme nnnn cctgagctcgag GoldenBraid makes use of type IIS restriction enzymes Combining traits is easy.

6. The Golden Gate Assembly System 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:

7. PART 1 PART 2 Lac Z pENTR_Part1pENTR_Part2 pDEST Combining traits is easy. Golden Gate functions with TypeIIS restriction enzymes

8. PART 1 PART 2 pDEST Seamless, multipartite assembly High efficiency Combinatorial One tube-one reaction: Ligase + RE (cycles 16ºC and 37ºC) Combining traits is easy. The Golden Gate Assembly System

9. pENTR_PROM pDEST pENTR_Term atggtgaactta ttgg LB RB Lac Z Golden Gate opened the way to multipartite assembly of standarized DNA pieces Combining traits is easy.

10. pEXPRESSION COLLECTION OF STANDARIZED PARTS 1-4 4-3 3-2 atggtgaactta ttgg LB RB Combining traits is easy.

11. pEXPRESSION END OF THE STORY… atggtgaactta ttgg LB RB Combining traits is easy. Golden Gate did not allow multigene assembly.

12. 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.

13. pEXPRESSION atggtgaactta ttgg END OF THE STORY…UNLESS LB RB Combining traits is easy.

14. pEXPRESSION atggtgaactta ttgg 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.

15. Lac Z GoldenBraid assembly pENTR_Part1 pDEST pENTR_Part1 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.

16. pDGB_C12B pDGB_1AB3 pDGB_A12C Level α plasmids Level Ω plasmids 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.

17. GoldenBraid takes multipartite Golden gate composite parts and assembly them binarely 16h to colonies!! P 1 P 2 P 3 TU1 TU2 P 1 P 2 P 3 GoldenBraid TU1 TU2 The GoldenBraid Assembly System Combining traits is easy.

18. GoldenBraid assembly Combining traits is easy.

19. pDGB_C12B pDGB_1AB3 pDGB_A12C The 4 pEDGBs give a doble-loop structure (braid) Combining traits is easy.

20. Each destination plasmid performs a different braid twist TU1 TU2 pDGB_1AB3 Combining traits is easy.

21. pDGB_A12C In each Braid twist 2 composite parts are assembled together TU3 TU1 TU2 Combining traits is easy.

22. GoldenBraid assemblies are reusable and ready to transform TU3 TU1 TU2 TU4 TU5 pDGB_1AB3 Combining traits is easy.

23. pDGB_1AB3 35S-RED-NT Red Fluorescent Protein 35S-BFP-NT Blue Fluorescent Protein 35S-P19-NT Silencing Supressor 35S-GFP-NT Green Fluorescent Protein Combining traits is easy: a real example

24. pDGB_1AB3 Only one day work up to 2X TU assembly!! 35S-RED-NT Red Fluorescent Protein 35S-BFP-NT Blue Fluorescent Protein 35S-P19-NT Silencing Supressor 35S-GFP-NT Green Fluorescent Protein Combining traits is easy: a real example

25. pDGB_A12C 35S-RED-NT Red Fluorescent Protein 35S-BFP-NT Blue Fluorescent Protein 35S-P19-NT Silencing Supressor 35S-GFP-NT Green Fluorescent Protein Combining traits is easy: a real example

26. + + + GB assembly ensures coordinated expression in N. benthamiana Combining traits is easy: a real example

27. + + + GB assembly ensures coordinated expression in N. benthamiana Combining traits is easy: a real example

28. 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.

29. Alejandro Sarrión-Perdigones Paloma Juárez Jose Manuel Julve Asun Fernández Silvia Presa Antonio Granell Diego Orzáez ¡GRACIAS! Fruit genomics and Biotechnology Group.