Team:Imperial College London/Project/Switch/Overview

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As part of our human practices work, we need to consider what will happen in the event that these bacteria are released into the soil. The potential consequences of their release relate to their uncontrolled spread and the possibility that they pass on the auxin genes to a potentially pathogenic bacterium.
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To help towards these issues, we have designed a few safety features that will go some of the way towards making this project safer.
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<h1>Specification - Preventing Horizontal Gene Transfer</h1>
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As part of our human practices work, we need to consider what will happen in the event that our modified bacteria are released into the soil. The potential consequences of their release relate to their uncontrolled spread and the possibility that they pass on the auxin genes to naturally occurring soil bacteria.</p>
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The auxin compound that we are using is the natural indole-3-acetic acid, which is not used as a herbicide like many other synthetic auxins. However, in high concentrations, indole-3-acetic acid can retard plant growth - as shown in our experiments.</p>
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While there are already a few species of bacteria that are able to secrete auxin<sup>[1]</sup>, it would be careless of us to release our bacteria without giving some thought to a containment device. So
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Kill Switch Designs
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<h2>The Gene Guard Design</h2>
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We have implemented the Holin/Anti-Holin regulated kill switch designed by the Berkeley 2008 iGEM team to create a system limiting horizontal gene transfer. Holin is a protein that forms pores in cell membranes and anti-holin binds to holin, inhibiting it's action. Once pores are formed by holin, lysozyme can access the periplasmic space and degrade the cell wall, causing cell lysis.</p>
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In the BacTrap system, the antiholin gene will be on the genome of our engineered bacteria under the control of a strong promoter. The Holin and Lysozyme genes will be present on the same plasmid as the two auxin genes and the chemoreceptor gene. The idea here is that the presence of the antiholin will prevent the cell from lysing from the effects of holin and lysozyme. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and lysozyme will kill the cell, preventing it from keeping the plasmid containing the auxin genes. This mechanism will prevent the succesfull horizontal gene transfer to naturally occurring soil bacteria.</p>
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One flaw in the design, is that, due to the time constraints placed upon us by the iGEM competition, the system that we will engineer will have antibiotic resistance genes on the genome. This is not something that is intended as part of the overall design, but is necessary for this competition.</p>
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The first idea was to implant a chemical into the selected region of soil that the bacteria would be unable to live without, but this was dismissed on environmental reasons as there is very little that we can add to the soil without damaging its composition or the organisms within.
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<p><embed src="https://static.igem.org/mediawiki/2011/4/48/ICL_GeneGuard.swf" width="935px" height="500px" /></p>
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The second idea was to implement the Holin/Anti-Holin regulated kill switch designed by the Berkeley 2008 iGEM team under the control of a UV sensitive promoter. Since UV light can only penetrate 0.3mm into soil, this would be an ideal method to ensure that the bacteria remain underground.
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<h2>Modelling - Informing the Design</h2>
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The key to the success of this design is the tight ratio between the amounts of holin and antiholin produced, and this needs to be carefully modelled before it can be assembled. The alternative method is to produce a promoter library using site-directed mutagenesis and then select for the survivors. This is, however, a time-consuming process and is extremely inefficient.
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By modelling the production
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The third idea uses the same BioBrick parts from the 2008 Berkeley iGEM team to create a toxin/anti-toxin system to try and limit horizontal gene transfer. The Antiholin gene will be on the genome of the bacteria under the control of a strong promoter. The Holin and Lysozyme genes will be present on the same plasmid as the two auxin genes. The idea here is that the presence of the antiholin will prevent the cell from lysing from the effects of holin and lysozyme. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and lysozyme will kill the cell, preventing it from keeping the plasmid containing the auxin genes.
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<h3>References</h3>
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[1] http://m.biotecharticles.com/Biology-Article/Natural-Growth-Hormone-IAA-Indole-3-Acetic-Acid-602.html
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Cloning Strategy
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The first step is to transform competent E.coli cells with the killswitch cassette from Berkeley 08, available in the registry. This will then give us many copies of the genes so that we can use them in transformations.
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Next, we will need to obtain a source of the antiholin gene by itself, and this will be achieved using PCR.
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The next, and quite possibly the hardest step will be to transform E.coli so that antiholin is present on the genome. For this, we will use versatile conditional-replication, integration, and modular (CRIM) plasmids.
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Latest revision as of 14:56, 13 September 2011



Specification - Preventing Horizontal Gene Transfer

As part of our human practices work, we need to consider what will happen in the event that our modified bacteria are released into the soil. The potential consequences of their release relate to their uncontrolled spread and the possibility that they pass on the auxin genes to naturally occurring soil bacteria.

The auxin compound that we are using is the natural indole-3-acetic acid, which is not used as a herbicide like many other synthetic auxins. However, in high concentrations, indole-3-acetic acid can retard plant growth - as shown in our experiments.

While there are already a few species of bacteria that are able to secrete auxin[1], it would be careless of us to release our bacteria without giving some thought to a containment device. So

The Gene Guard Design

We have implemented the Holin/Anti-Holin regulated kill switch designed by the Berkeley 2008 iGEM team to create a system limiting horizontal gene transfer. Holin is a protein that forms pores in cell membranes and anti-holin binds to holin, inhibiting it's action. Once pores are formed by holin, lysozyme can access the periplasmic space and degrade the cell wall, causing cell lysis.

In the BacTrap system, the antiholin gene will be on the genome of our engineered bacteria under the control of a strong promoter. The Holin and Lysozyme genes will be present on the same plasmid as the two auxin genes and the chemoreceptor gene. The idea here is that the presence of the antiholin will prevent the cell from lysing from the effects of holin and lysozyme. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and lysozyme will kill the cell, preventing it from keeping the plasmid containing the auxin genes. This mechanism will prevent the succesfull horizontal gene transfer to naturally occurring soil bacteria.

One flaw in the design, is that, due to the time constraints placed upon us by the iGEM competition, the system that we will engineer will have antibiotic resistance genes on the genome. This is not something that is intended as part of the overall design, but is necessary for this competition.

Modelling - Informing the Design

The key to the success of this design is the tight ratio between the amounts of holin and antiholin produced, and this needs to be carefully modelled before it can be assembled. The alternative method is to produce a promoter library using site-directed mutagenesis and then select for the survivors. This is, however, a time-consuming process and is extremely inefficient.

By modelling the production

References

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

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