Team:Imperial College London/Project Gene Design

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<h1>Design</h1>
<h1>Design</h1>
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<p>We aim to ensure that the <a href="https://2011.igem.org/Team:Imperial_College_London/Project_Gene_Specifications"><b>specifications</b></a> that were drawn up are considered in the design of Gene Guard.
<p><b>1. Prevent horizontal gene transfer by making any other cell that is not our own GMO non-viable</b></p>
<p><b>1. Prevent horizontal gene transfer by making any other cell that is not our own GMO non-viable</b></p>
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<ul class="a">
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<p>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.</p>
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<li><p> T4 endolysin and T4 holin can be inverse PCR'd from <a href="http://partsregistry.org/Part:BBa_K112808"><b>BBa_K112808</b></a>. We must, however, ensure that the cells that receive these genes will lyse. In order to determine which promoter to use, 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 <a href="http://partsregistry.org/Part:BBa_J23103"><b>J23103</b></a> promoter had the correct strength relative to the <a href="http://partsregistry.org/Part:BBa_J23100"><b>J23100</b></a> 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.</li></p>
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</ul>
<p><b>2. Our own GMO must not be harmed by this module</b></p>
<p><b>2. Our own GMO must not be harmed by this module</b></p>
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<ul class="a">
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<li><p>This module relied heavily on modelling during the design process. We had to take into account the fact that plasmid copy number and copy number in the genome are variable when designing the toxin and anti-toxin components. We found that the promoter of the holin-endolysin plasmid has to be 40-400 times weaker than that of the holin construct in the genome to be effective. This ratio should take the variability between the gene copy number into account and ensure that there is at least one holin molecule for every anti-holin molecule produced.</li></p>
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<p>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 deciding the two components.
 
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<p><b>3. Must not be too much of a metabolic burden</b></p>
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<li><p>To be sure that our modified bacteria will survive holin and endolysin production, we chose the <a href="http://partsregistry.org/Part:BBa_J23103"><b>J23103</b></a> promoter which has a strength at the lower end of promoter strength range. This provided the design of the following two constructs:</li></p>
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<p><b>4. Must be able to test whether it works</b></p>
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</ul>
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<p><b>3. Expression of the device must not be too much of a metabolic burden</b></p>
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<ul class="a">
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<li><p>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, AuxIn is a proof of concept system. Once it has been shown that Gene Guard is a viable containment method, we will modify the construct in order to achieve the ideal balance between having the anti-holin inactivate holin, while not being a large burden on the cell.</li></p>
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</ul>
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<p><b>4. Being able to test if the system works </b></p>
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<ul class="a">
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<li><p> In order to do this we decided to attach an RFP coding sequence under the same promoter as the holin and endolysin genes. This will allow us to easily and visually test whether the cells contain our plasmid. As for the holin construct, the CRIM plasmid already contains a sfGFP sequence.</li></p>
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<li><p>We will be able to distinguish our completed construct from every other cell due to its kanamycin and ampicillin resistance as well as its production of both RFP and sfGFP. Therefore, we will be able to see the transfer of our plasmid into a cell that does not fluoresce green and should be able to track whether it lyses or not under a wide-field microscope.</li></p>
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</ul>
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<p>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.</p>
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<h2>Bibliography</h2>
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<p>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<sup>[1]</sup></p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>In order to integrate the antiholin gene into the genome, we will use a CRIM plasmid <sup>[2]</sup>. 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.</p>
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<h2>References</h2>
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<p>[1] Gründling et al. (2001) Holins kill without warning. <i>PNAS</i> <b>98(16):</b> 9348-9352</p>
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<p>[1] - 1] Gründling et al.. (2001). Holins kill without warning. PNAS. 16 </p>
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<h2>
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<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Gene_Specifications" style="text-decoration:none;color:#728F1D;float:left;">
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M3: Specification
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<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Gene_Modelling" style="text-decoration:none;color:#728F1D;float:right;">
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M3: Modelling
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Latest revision as of 23:38, 16 October 2011



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

We aim to ensure that the specifications that were drawn up are considered in the design of Gene Guard.

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

  • T4 endolysin and T4 holin can be inverse PCR'd from BBa_K112808. We must, however, ensure that the cells that receive these genes will lyse. In order to determine which promoter to use, 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. We had to take into account the fact that plasmid copy number and copy number in the genome are variable when designing the toxin and anti-toxin components. We found that the promoter of the holin-endolysin plasmid has to be 40-400 times weaker than that of the holin construct in the genome to be effective. This ratio should take the variability between the gene copy number into account and ensure that there is at least one holin molecule for every anti-holin molecule produced.

  • To be sure that our modified bacteria will survive holin and endolysin production, we chose the J23103 promoter which has a strength at the lower end of promoter strength range. This provided the design of the following two constructs:

3. Expression of the device 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, AuxIn is a proof of concept system. Once it has been shown that Gene Guard is a viable containment method, we will modify the construct in order to achieve the ideal balance between having the anti-holin inactivate holin, while not being a large burden on the cell.

4. Being able to test if the system works

  • In order to do this we decided to attach an RFP coding sequence under the same promoter as the holin and endolysin genes. This will allow us to easily and visually test whether the cells contain our plasmid. As for the holin construct, the CRIM plasmid already contains a sfGFP sequence.

  • We will be able to distinguish our completed construct from every other cell due to its kanamycin and ampicillin resistance as well as its production of both RFP and sfGFP. Therefore, we will be able to see the transfer of our plasmid into a cell that does not fluoresce green and should be able to track whether it lyses or not under a wide-field microscope.

Bibliography

[1] Gründling et al. (2001) Holins kill without warning. PNAS 98(16): 9348-9352

M3: Specification M3: Modelling



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