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From 2011.igem.org

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Module 3: Gene Guard

Containment is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent horizontal gene transfer of the genes we are expressing in our chassis, we have developed a system based on the genes encoding holin, anti-holin and endolysin. We are engineering anti-holin into the genome of our chassis, where it acts as an anti-toxin, and holin and endolysin on plasmid DNA. In the event of horizontal gene transfer with a soil bacterium, holin and endolysin will be transferred without anti-holin, rendering the recipient cell non-viable and effectively containing the Auxin Xpress and Phyto-Route genes in our chassis.

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Design

We have implemented the Holin/Anti-Holin regulated kill switch designed by the Berkeley 2008 iGEM team to create a system limiting horizontal gene transfer. Holin is a protein that forms pores in cell membranes and anti-holin binds to holin, inhibiting it's action. Once pores are formed by holin, endolysin can access the periplasmic space and degrade the cell wall, causing cell lysis.

In the GeneGuard system, the antiholin gene will be on the genome of our engineered bacteria under the control of a strong promoter. The Holin and Endolysin genes will be present on the same plasmid as the two auxin genes and the chemoreceptor gene. The idea here is that the presence of the antiholin will prevent the cell from lysing from the effects of holin and endolysin. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and endolysin will kill the cell, preventing it from keeping the plasmid containing the auxin genes. This mechanism will prevent the succesful horizontal gene transfer to naturally occurring soil bacteria.

This will require a careful balance of the levels of expression of holin and antiholin, although it is better to have an increased expression of antiholin to provide some protection to our cells. The expression levels will be governed by the promoter and RBS combination. In order to make this easier, we fixed the promoter in front of the antiholin as J23100, and then the Salis Lab RBS designer was used to generate an RBS sequence that would give the correct level of expression, a value that would be generated using computer modelling.

Because we have to use the holin gene that is in the cell lysis cassette submitted to the registry by Berkeley 2008, it would be too difficult to replace the RBS that is upstream of the gene. Instead, we calculated its strength using the Salis Lab RBS calculator and used this as a fixed value around which to model the required promoter strength.

As can be seen from the data presented on the modelling page, we were able to calculate the required RBS strength for the holin expression and the appropriate promoter for the anitholin expression. Our RBS

One flaw in the design, is that, due to the time constraints placed upon us by the iGEM competition, the system that we will engineer will have antibiotic resistance genes on the genome. This is not something that is intended as part of the overall design, but is necessary for this competition.