Team:Imperial College London/Project Gene Design

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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.



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 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 came with an sfGFP sequence.

To create a system to prevent horizontal gene transfer of our genetic constructs, we designed a toxin-antitoxin system. We will be using the T4 Anti-Holin as the anti-toxin and the T4 Holin and endolysin as the toxin.

Holin is a protein that forms pores in cell membranes and anti-holin binds to holin, inhibiting its action. Once pores are formed by holin, endolysin can access the periplasmic space and degrade the cell wall, causing cell lysis[1]

Our chassis will have the gene for antiholin on its genome, and holin and endolysin on the plasmid with the auxin genes. This would mean that while the auxin plasmid is inside our chassis, antiholin would inhibit the activity of holin and prevent lysis. However, when that same plasmid is passed on to another bacterium in the soil by conjugation, the recipient bacterium will not have antiholin on its genome and so the holin concentration will build up. As the holin builds up inside the cell, it will form pores in the inner membrane that will allow the endolysin to enter the periplasmic space and break down the cell wall, causing the cell to lyse.

So it can be seen that the crucial part of our system is the balance between the levels of holin and antiholin in the cell. This will require careful modelling to influence our design, as the holin level should be sufficiently high as to induce lysis quickly in other cells, but low enough as to be easily controllable inside our own chassis.

The expression levels will be governed by the promoter and RBS combination. In order to make this easier, we fixed the promoter in front of the antiholin as J23100, and then the Salis Lab RBS designer was used to generate an RBS sequence that would give the correct level of expression, a value that would be generated using computer modelling.

Because we are limited to using the antiholin gene that is in the cell lysis cassette submitted to the registry by Berkeley 2008, it would be too difficult to replace the RBS that is upstream of the gene. Instead, we calculated its strength using the Salis Lab RBS calculator and used this as a fixed value around which to model the required promoter strength.

As can be seen from the data presented on the modelling page, we were able to calculate the required RBS strength for the antiholin expression and the appropriate promoter for the holin expression. Our RBS was generated by the Salis Lab RBS designer to give a level of expression that is stronger than necessary to ensure that our bacteria are protected. The promoter for the holin was chosen from the Anderson promoter library, using the relative promoter strengths as a guide. We chose J23103 because it is one of the weakest promoters in the Anderson library.

In order to integrate the antiholin gene into the genome, we will use a CRIM plasmid [2]. This will however, introduce a kanamycin resistance gene into the genome of our bacteria, which is far from ideal in a project that is designed to be released into the environment. This is not something that is intended as part of the overall design, but is necessary due to the time constraints in the iGEM competition.

References

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

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