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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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Future Work

To carry on the work on the Gene Guard module, there are a number of steps we would take in the immediate future and others that form part of our long term plan.

Short-term plans

1. Assembly of Gene Guard

We are planning to fully assemble Gene Guard. We have already expressed anti-holin in our cells and will be working towards assembling the entire construct.

2. 16s rRNA of the bacteria grown up from sterile and non-sterile soil will be sequenced to determine the bacterial species present.

This sequencing will allow us to confirm that the bacteria we were able to rescue from soil are indeed E. coli. This will hopefully confirm that our bacteria have been able to retain their plasmids for over a month while exposed to competition from other soil microorganisms.

3. Replicates of the soil experiment will be analysed for expression of sfGFP.

This will allow us to establish statistical significance of our results. At present, we need to test replicates for the presence and functionality of the GFP gene.

4. Observing Gene Guard in action

We will investigate how to induce conjugation in bacteria. We will use the RFP incorporated in the Gene Guard construct to image transferral of RFP to other bacteria prior to lysis on a widefield microscope.

Long-term plans

1. Alternative to antibiotic resistance

During the assembly and testing of this module, we have used antibiotic resistance genes to select for bacteria expressing the desired genes. However, we will not be able to release bacteria with antibiotic resistance genes into field trials. We are therefore planning to use already existing toxin/anti-toxin systems to select for genes.

2.Testing and implementation in the field.

We are going to develop plans of how we would be able to conduct field trials of Gene Guard. These trials will be necessary for ensuring the safety and efficacy of our construct.