Golden Braid A Standarized DNA Assembly System for

"Golden. Braid: 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. Ideally, plant genetic parts and modules are better assembled at DNA level Ideal features of a DNA assembly system for Plant Biotechnology: -Standarized. -Reusable pieces and modules. -Simple (rules and toolkit). -Efficient

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

Combining traits is easy. Golden. Braid makes use of type IIS restriction enzymes ctcgag Type II enzyme nnnn cctgag Type IIS enzyme

Combining traits is easy. Golden. Braid is based on Golden Gate, an assembly system described previously: The Golden Gate Assembly System One tube-one reaction Golden Gate is a extreamly efficient, potentialy seamless multipartite assembly system Engler C, et al (2009) PLo. S One 4: e 5553. Engler C, Kandzia R, Marillonnet S (2008) PLo. S One 3: e 3647.

Combining traits is easy. Golden Gate functions with Type. IIS restriction enzymes p. ENTR_Part 1 Bsa. I 1 p. ENTR_Part 2 3 Bsa. I PART 1 1 Bsa. I Lac Z p. DEST Bsa. I 3 2 PART 2 2 Bsa. I

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

Combining traits is easy. Golden Gate opened the way to multipartite assembly of standarized DNA pieces p. ENTR_Term Bsa. I 4 CDS 3 Bsa. I p. ENTR_Term p. ENTR_PROM Bsa. I 1 4 PROM ctta 1 1 LB Bsa. I atgg PROM Bsa. I 4 3 tgaa CDS Lac Z p. DEST 3 TERM 2 Bsa. I ttgg TERM Bsa. I 2 2 RB

Combining traits is easy. COLLECTION OF STANDARIZED PARTS 4 -3 1 -4 3 -2 ctta 1 atgg PROM 4 tgaa CDS LB p. EXPRESSION 3 TERM ttgg 2 RB

Combining traits is easy. END OF THE STORY… Golden Gate did not allow multigene assembly. ctta 1 LB atgg PROM 4 tgaa CDS p. EXPRESSION 3 TERM ttgg 2 RB

Combining traits is easy. ACTUALLY, IT WAS THE START OF THE STORY… The Golden. Braid Assembly System Sarrión-Perdigones et al (2011) Plo. S One 6(7): e 21622.

Combining traits is easy. END OF THE STORY…UNLESS ctta Bsb. I LB A 1 atgg PROM 4 tgaa CDS p. EXPRESSION 3 TERM ttgg 2 C Bsb. I 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. TRANSCRIPTIONAL UNIT 1 ctta Bsb. I LB A 1 atgg PROM 4 tgaa CDS p. EXPRESSION 3 TERM ttgg 2 C Bsb. I RB

Combining traits is easy. Golden. Braid assembly p. ENTR_Part 1 Bsb. I A TU 1 p. ENTR_Part 1 C A Bsb. I Lac Z Bsb. I C TU 2 B Bsb. I B p. DEST 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. Golden. Braid uses a simple toolbox of 4 destination plasmids (p. DGBs) A 1 Z 2 C p. DGB_A 12 C To solve this dead end we developed Golden. Braid, an iterative system that allows indefinite assembly of new TUs. Level α plasmids C 1 Z 2 B p. DGB_C 12 B 1 AZB 3 p. DGB_1 AB 3 Level Ω plasmids 3 AZB 2 p. DGB_1 AB 3

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

Golden. Braid assembly Combining traits is easy.

Combining traits is easy. The 4 p. EDGBs give a doble-loop structure (braid) A 1 Z 2 C p. DGB_A 12 C C 1 Z 2 B p. DGB_C 12 B 1 AZB 3 p. DGB_1 AB 3 3 AZB 2 p. DGB_1 AB 3

Combining traits is easy. Each destination plasmid performs a different “braid twist” 1 AZB 3 p. DGB_1 AB 3 TU 1 A C TU 2 C B 1 3

Combining traits is easy. In each “Braid twist” 2 composite parts are assembled together A 1 Z 2 C p. DGB_A 12 C A C TU 1 1 3 TU 2 TU 3 2 3

Combining traits is easy. Golden. Braid assemblies are “reusable” and “ready to transform” TU 1 TU 2 TU 3 TU 4 TU 5 3 AZB 2 p. DGB_1 AB 3

Combining traits is easy: a real example Silencing Supressor Green Fluorescent Protein A G C C 35 S-GFP-NT P B 35 S-P 19 -NT Blue Fluorescent Protein C B B 35 S-BFP-NT Red Fluorescent Protein A R C 35 S-RED-NT 1 AZB 3 p. DGB_1 AB 3 A G C C B B 1 G B 3

Combining traits is easy: a real example Green Fluorescent Protein A G C Silencing Supressor C 35 S-GFP-NT P B 35 S-P 19 -NT Blue Fluorescent Protein C B Red Fluorescent Protein A B 35 S-BFP-NT R C 35 S-RED-NT Only one day work up to 2 X TU assembly!! A C R C P B 1 3 3 AZB 2 p. DGB_1 AB 3 G B 3 R P 2

Combining traits is easy: a real example Green Fluorescent Protein A G C Silencing Supressor C 35 S-GFP-NT P B 35 S-P 19 -NT Blue Fluorescent Protein C B B 35 S-BFP-NT Red Fluorescent Protein A R C 35 S-RED-NT A 1 Z 2 C p. DGB_A 12 C A G B R P C 1 3 G B 3 R P 2

Combining traits is easy: a real example GB assembly ensures coordinated expression in N. benthamiana A G A R C + C B B B + R A P C R C + C P B

Combining traits is easy: a real example GB assembly ensures coordinated expression in N. benthamiana A G A R C + C B B B + R A P C R C + C P B

Combining traits is easy. Golden. Braid 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.

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