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 BsmBIor 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

Sticky ends:

As we were using GoldenGate cloning, we had to design parts with sticky ends that are complementary, and so would ligate into the correct order. Seeing as we used some parts that were already in the registry, we had to design sticky ends that had not been used before. Typically, teams such as the Cardiff team from 2017, would use only 3 parts in level 1 constructs. These would usually be a promoter, coding sequence, and terminator. However, due to synthesis size limits, we had to create constructs using up to 5 level 0 parts. Whilst GoldenGate cloning reports that around 11 level 0 parts can be combined at once, it means that for each order a different set of sticky ends must be created and used. For example, the most 5' sequence of our constructs was the promoter, with the 5' sticky end having GAGG to ligate to the plasmid backbone, and its 3' end having TACT to ligate to the region of DNA to be second in position. In our case, this was either the OTMV enhancer, or the 5' sequence of our siRNAs. Visit our individual part pages to find out all the sequences.

From BsmBI to Esp3I:

Early on in the project, we found difficulty running the level 0 digestion and ligation reactions simultaneously due to the optimum temperature of the restriction endonuclease. Typically, GoldenGate reactions are with digestion and ligation occurring in the same PCR tube, running in a thermocycler. However, initially we were using the type IIS restriction enzyme, BsmBI, which has an optimum temperature of 55 degrees Celsius. Running a cycled reaction with T4 DNA ligase (with an optimum temperature of 16 degrees Celsius and a denaturation temperature of around 65 degrees Celsius) raised some red flags. We found our reaction efficiency to be very low, with some results running the two reactions separately, but much less efficiently. One team member discovered that BsmBI has an isoschizomer, a restriction endonuclease that cuts at the same region, called Esp3I that has an optimum temperature of 37 degrees Celsius, well within the temperature range that allows T4 ligase to maintain is form. Once we were using this enzyme for our reactions, nearly all of them worked first time, with nearly 100% of colonies appearing white after an overnight cycle.

Changes to GUS reagents:

Later on in the project, we found that our GUS staining solution assay was not giving the desired results. One of the researchers in the university, Dr. Scofield, allowed us to use some of his GUS staining solution, and gave us his modified protocol for GUS staining. We immediately found clearer and stronger GUS staining.

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.

What should this page contain?

Explanation of the engineering principles your team used in your design
Discussion of the design iterations your team went through
Experimental plan to test your designs

Team:Cardiff Wales/Design

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