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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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Specifications

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

• Horizontal gene transfer (Figure 1) was the main problem that we could think of when it came to releasing GMOs in field tests and later in the implementation stage of the project. This is a common issue even in GM crops where cross-pollination between the GMO and natural organisms is a constant 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 and induce a response in the naturally occurring bacterium instead.

• Looking into kill switches that have been used in the past, we 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.

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. The CRIM (conditional-replication, integration, and modular) plasmid can be integrated in single copies into the genome of E. coli^[2].

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

• Since our final system will be using the Phyto-Route and Auxin Xpress modules we had to consider that the modified bacteria would already be under a huge metabolic burden. Therefore, a Gene Guard that uses the minimal resources necessary would be beneficial to promote chemotaxis towards malate and IAA production by our system.

4. Must be able to test whether the system works

• To do this we must use reporter genes. The most common reporter genes are fluorescent proteins like RFP and GFP. Since the CRIM plasmid already has sfGFP, we will be using that to test for the expression of anti-holin. In order to test whether the plasmid is functional, we will be integrating RFP into the plasmid. We hope to be able to see a natural bacterium that has taken up the plasmid fluoresce red before lysing by widefield microscopy.

References

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

[2] Haldimann and Wanner (2001) Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. Journal of Bacteriology 183(21): 6384-6393.

M3: Overview M3: Design