Design

Principles

Being one of the few iGEM teams that makes transgenic plants as part of our project, we needed to take into consideration the extra steps required to make genetically modified plants. More specifically, the transformation procedure was both modular and sequential, requiring transformation of E. coli, then Agrobacterium tumefaciens, followed by infiltration into Nicotiana benthamiana.

GoldenGate cloning:

As a team, we decided to use the efficient assembly allowed by the GoldenGate system, creating a range of parts that are assembled in a specific order. This relies on using Type IIS restriction enzymes that cut downstream of a recognition site and create sticky ends of the designer's choice. This allows a range of custom sticky ends to be created to allow the effective, clean ligation of different DNA sequences, with minimal additional DNA. This system requires the creation of DNA sequences that contain the BsmB1or Esp3Irecognition sites 5'- CGTCTC(N)₁ | -3' and 3'- GCAGAG(N)₅ | -5', where "|" is the site of endonuclease activity. For our project, these sequences were ordered as gBlocks from IDT. These were then set up in a simultaneous digest/ligation reaction to insert these parts into the level 0 acceptor plasmid, and iGEM plasmid backbone, pSB1C3. These sticky ends are created so that when the level 0 parts ligate into the pSB1C3 plasmid, the cut sites for another Type IIS restriction endonuclease, BsaI,is created. These are the sequences 5' - GGTCTC(N)₁ | -3' and 3'- CCAGAG(N)₅ | -5'. Following this, a combined reaction with several level 0 parts can be set up that, due to their custom sticky ends, can be simultaneously ligated into one specific order with very high efficiency. Following each digestion/ligation reaction, E. coli would be transformed and grown overnight on antibiotic containing plates.

Bacterial selection:

To detect which bacterial colonies had the desired insert, we used both negative and positive selection. Bacteria that had not taken up the plasmid, be it pSB1C3 or pGBA2 for a level 0 and level 1 reaction, respectively, would be killed by the antibiotic in the plates (either chloramphenicol for level 0 plates, or kanamycin for level 1 plates). The resulting colonies on the plates would either be blue, containing the original LacZ gene in the acceptor plasmid which breaks down X-gal, or white, where LacZ had been lost, so X-gal is not cleaved, and the colony remains colourless. Consequently, white colonies would be screened by colony PCR to detect the presence of the insert. Colonies identified as promising were then picked, grown in antibiotic-containing broth, and had plasmid DNA extracted for DNA sequencing. Following this, Agrobacterium tumefaciens were transformed with sequence-confirmed DNA, and grown on plates containing kanamycin and rifamycin.

Plant transformation:

A. tumefaciens colonies were subsequently picked, activated, and syringe-infiltrated by hand into around 50 day old Nicotiana benthamiana plants. We found the best transgene expression measuring reporter gene output 4 days post infiltration (DPI), following a heat shock at 37 degrees Celsius for 30 minutes at 2DPI. For more detailed, replicable protocols, please visit our protocols page.

Iterations

Design iterations being sticky ends and overlapping sites, changing BsmBI for Esp3I. Changes to GUS reagents.

Experimental design

Tests for GFP and mCherry in the imaging room. Test for siRNA expression using semi-quantitative PCR. Test assay for GUS. Assay for the Ribitol promoter collaboration for WashU.

Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.

This page is different to the "Applied Design Award" page. Please see the Applied Design page for more information on how to compete for that award.

• Discuss GoldenGate cloning, blue/white selection, the use of E. coli, Agrobacterium, and then tobacco
• Design iterations being sticky ends and overlapping sites, changing BsmBI for Esp3I. Changes to GUS reagents.
• Tests for GFP and mCherry in the imaging room. Test for siRNA expression using semi-quantitative PCR. Test assay for GUS. Assay for the Ribitol promoter collaboration for WashU.