Team:Imperial College London/Project/Switch/Overview

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<h1>Containment Device</h1>
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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 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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The auxin 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 growth - as shown in our experiments.  
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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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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.
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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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Of course, that is a very worst case scenario, but it highlights one of the main problems within synthetic biology: We do not know what will happen when they are released, and we cannot find out without releasing them.
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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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<h2>The Gene Guard Design</h2>
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<h2>Kill Switch Designs</h2>
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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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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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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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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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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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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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<p><embed src="https://static.igem.org/mediawiki/2011/4/48/ICL_GeneGuard.swf" width="935px" height="500px" /></p>
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<h2>Modelling - Informing the Design</h2>
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<h2>Cloning Strategy</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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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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By modelling the production
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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. The CRIM plasmid is designed so that it integrates into a specific site on the genome, allowing us to ensure that there is only one copy of the gene on the genome.
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<h2>Promoter Design</h2>
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The key part of this module is the need for the amount of Antiholin to be equal to the amount of Holin so that the auxin-secreting cells are not lysed. However, the promoter needs to be strong enough to allow enough holin/lysozyme production so that the recipient cell will be lysed quickly.
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Our first model assumed that the genes on the plasmids would be expressed at around 100-300 times the level of expression from the genome, because of the high copy number plasmid we were planning to use. However, after consulting with Tom Ellis, we had to scrap that model.
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In a previous project comparing the levels of expression of GFP on the genome of E.coli against the same gene on a high copy number plasmid, the difference in the expression levels were calculated using the cell's fluorescence. The difference was a mere 7%.
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<h3>References</h3>
<h3>References</h3>
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[1] http://m.biotecharticles.com/Biology-Article/Natural-Growth-Hormone-IAA-Indole-3-Acetic-Acid-602.html
[1] http://m.biotecharticles.com/Biology-Article/Natural-Growth-Hormone-IAA-Indole-3-Acetic-Acid-602.html
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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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