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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 aim to ensure that the specifications that were drawn up are considered in the design of Gene Guard.

1. Prevent horizontal gene transfer by making any other cell that is not our own GMO non-viable

• T4 endolysin and T4 holin can be inverse PCR'd from BBa_K112808. We must, however, ensure that the cells that receive these genes will lyse. In order to determine which promoter to use, we modelled the entire system. Since there are so many copies of the holin and endolysin (high copy plasmid) and so few copies of the holin gene (genome) we decided that the J23103 promoter had the correct strength relative to the J23100 promoter we chose. However, we also had to model whether this weak promoter would be enough to lyse the cell that receives the plasmid. According to our modelling, any cell that receives our holin and endolysin genes will lyse.

2. Our own GMO must not be harmed by this module

• This module relied heavily on modelling during the design process. We had to take into account the fact that plasmid copy number and copy number in the genome are variable when designing the toxin and anti-toxin components. We found that the promoter of the holin-endolysin plasmid has to be 40-400 times weaker than that of the holin construct in the genome to be effective. This ratio should take the variability between the gene copy number into account and ensure that there is at least one holin molecule for every anti-holin molecule produced.

• To be sure that our modified bacteria will survive holin and endolysin production, we chose the J23103 promoter which has a strength at the lower end of promoter strength range. This provided the design of the following two constructs:

3. Expression of the device must not be too much of a metabolic burden

• While this specification is important, it did not play a huge role in the design of this version of the module. This is because, for now, AuxIn is a proof of concept system. Once it has been shown that Gene Guard is a viable containment method, we will modify the construct in order to achieve the ideal balance between having the anti-holin inactivate holin, while not being a large burden on the cell.

4. Being able to test if the system works

• In order to do this we decided to attach an RFP coding sequence under the same promoter as the holin and endolysin genes. This will allow us to easily and visually test whether the cells contain our plasmid. As for the holin construct, the CRIM plasmid already contains a sfGFP sequence.

• We will be able to distinguish our completed construct from every other cell due to its kanamycin and ampicillin resistance as well as its production of both RFP and sfGFP. Therefore, we will be able to see the transfer of our plasmid into a cell that does not fluoresce green and should be able to track whether it lyses or not under a wide-field microscope.

Bibliography

[1] Gründling et al. (2001) Holins kill without warning. PNAS 98(16): 9348-9352

M3: Specification M3: Modelling