"

From 2011.igem.org

---

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.

---

Design

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

In order to design a system that will make other cells non-viable, we will have to design a plasmid that will contain T4 endolysin and T4 holin. These genes can be PCR'd out from BBa_K112808. We must, however make sure that the cells that receive these genes will lyse. In order to determine which promoter we used 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. Since plasmid copy numbers and copy numbers in the genome are variable we had to take this into account when designing the toxin and anti-toxin components. We discovered that the promoter of the holin-endolysin plasmid has to be 40-400 times weaker than that of the holin construct in the genome. This ratio should take the variability between the gene copy numbers into account and make it so that there is at least one holin molecule for every anti-holin molecule that is produced.

In order to assure that the cell will survive the holin and E endolysin we decided to use the lower end of the promoter strength range and chose J23103 as our promoter. This gave us the design of the following two constructs:

3. 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, this is a proof of concept. Once it has been shown that the Gene Guard is viable we will attempt to modify this construct in order to achieve the ideal balance between having the two components cancel each other while not being a large burden on the cell.

4. Must be able to test whether it works

Since this module is tackling a complex issue, we will need to design the constructs while keeping in mind that we will need to test whether it works once it is assembled. In order to do that we decided to attach an RFP coding sequence under the same promoter as the holin and endolysin genes. This will allow us to easily test whether the cells contain our plasmid. As for the holin construct, the CRIM plasmid already contains a sfGFP sequence.

Since 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.

References

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