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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, lysozyme 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 Lysozyme 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 lysozyme. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and lysozyme will kill the cell, preventing it from keeping the plasmid containing the auxin genes. This mechanism will prevent the succesfull horizontal gene transfer to naturally occurring soil bacteria.

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.