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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.

Specifications

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

Horizontal gene transfer was the main problem that we could think of when it came to releasing GMOs for field tests. This is a common issue even in GM crops where cross-pollination between the GMO and natural organisms is always a worry. In bacteria, genes are usually transferred to other species through conjugation or by the uptake of the genetic material from lysed GMOs. Since the genetic material can still be taken up by other species after the lysis of our GMO, a traditional kill switch where one uses an input to induce lysis is not a viable and safe option. Therefore, we thought that the device must be present within the genetic material itself.

We then looked at past kill switches to research what parts had been used previously and found the T4 holin-endolysin system. If we were to add these genes into our plasmid we could cause the immediate lysis of any organism that had taken up our plasmid.

Figure 1. Horizontal gene transfer (Grace Kim, 2006)

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

The T4 bacteriophage must be able to delay the lysis of its host before it is able to replicate and assemble many copies of itself. In order to do that the T4 bacteriophage also has a protein called anti-holin that binds to the holin thereby preventing the lysis of the cell. In essence, this system works as a timer giving the phage enough time to replicate.

By using the anti-holin from this system we could prevent our own cells from lysing. However, we cannot have it on a plasmid or else there might still be the possibility of an organism taking up both plasmids. Therefore we must anchor it within the genome. In order to do this we will need to use the CRIM plasmid.

3. Must not be too much of a metabolic burden

Since our final system would be using the Auxin Xpress and Phyto-Route modules we had to consider that the cells would already be under a huge metabolic burden. Therefore, a Gene Guard that uses the minimal resources necessary would be beneficial to IAA production in our system.

4. Must be able to test whether it works

In order to test whether our system works we must use reporter genes. The most common reporter genes are fluorescent proteins like RFP and GFP. Since the CRIM plasmid already has sfGFP in it we will be using that to test for the expression of the anti-holin aspect of the system. In order to test whether the plasmid works we will be adding RFP into the plasmid. We believe that we might able to see a cell fluoresce red before lysing under a widefield microscope.

References

Grace Kim, Aug 11, 2006. Figure 1. Horizontal gene transfer.[diagram] Available at: <http://www.scq.ubc.ca/attack-of-the-superbugs-antibiotic-resistance/> [Accessed 19 September 2011]

[1] http://m.biotecharticles.com/Biology-Article/Natural-Growth-Hormone-IAA-Indole-3-Acetic-Acid-602.html