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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 integrated anti-holin into the genome of 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

In order to quickly test whether the Gene Guard system works, we will try to transform a control cell line of DH10B cells with sfGFP in the genome and our Gene Guard cell line (sfGFP and anti-holin in the genome) with the holin-endolysin construct as well as the same construct without the holin-endolysin. Ideally, we would see no cells on the control and cells on the Gene Guard cell line plate.

Long-term plans

1. Horizontal gene transfer experiment

Since it is difficult to obtain lab strains of E. coli that contain the F plasmid (due to safety reasons) and can therefore conjugate we had to develop another experiment.

For this experiment, we would like to mix 100,000 to 1,000,000 Gene Guard cells with the holin-endolysin construct with MG1655 cells. Then we would grow them in a culture where the concentration of Ampicillin or Carbenicillin is kept at around 1 μg/ml for several hours to stress the cells. There is a very low probability that one of the cells will lose a plasmid and the MG1655 cells will pick it up. In order to look for this cell line (cells that have taken up the holin-endolysin plasmid) we would observe the cells using flow cytometry and taking advantage of the two fluorescent proteins involved. We would then also repeat the same experiment with the control cell line containing sfGFP in the genome and RFP on the plasmid.

2. 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 such as Delphi genetic's CCDB system (however, we would have to pay for the license to use it).

3. 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.

4. Investigating optimal promoters and RBSs for different wild type bacteria

The modelling tool will be used to establish a platform that defines optimal holin and anti-holin promoters and RBSs for different wild type bacteria.

M3: Testing & Results