Team:Dundee/Solution
From 2012.igem.org
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- | <li class='has-sub'><a href='https://2012.igem.org/Team:Dundee/ | + | <li class='has-sub'><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Wet Lab</span></a> |
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<li><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Strategy</span></a></li> | <li><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Strategy</span></a></li> | ||
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- | <li class='has-sub'><a href=' | + | <li class='has-sub'><a href='#'><span>Human Practices</span></a> |
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<li><a href='https://2012.igem.org/Team:Dundee/Safety'><span>Safety</span></a></li> | <li><a href='https://2012.igem.org/Team:Dundee/Safety'><span>Safety</span></a></li> | ||
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- | Continuing with the idea of reintroducing the natural gut bacteria to the colon | + | Continuing with the idea of reintroducing the natural gut bacteria to the colon in order to combat <I>Clostridium difficile</I> (<i>C. difficile</I>), our project aims to engineer a harmless <i>Escherichia coli</i> (<i>E. coli</i>) strain to target and kill <i>C. difficile</I> by expressing a specific endolysin. To do this, we will take a type VI secretion system (T6SS) from <I>Salmonella typhimurium LT2</I> and express it in <I>E. coli</I>. In addition, we will take the <I>C. difficile</I> -specific endolysin gene from a bacteriophage and fuse it with genes from the T6SS that are universally secreted. In order to control the system, we will incorporate a synthetic inflammation biosensor which will ensure that the system is only activated during times of infection. |
<br><br><center><img src="https://static.igem.org/mediawiki/2012/5/55/Solutionpic3.jpg"><br> | <br><br><center><img src="https://static.igem.org/mediawiki/2012/5/55/Solutionpic3.jpg"><br> | ||
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- | The endolysin we have chosen to use is from ΦCD27. This has been shown by Mayer et. al. to specifically kill 30 strains of <i>C. difficile</I>. We will aim to isolate the gene and fuse it to <i>Hcp</i> and <i>VgrG</i> genes which have been cloned | + | The endolysin we have chosen to use is from ΦCD27. This has been shown by Mayer et. al. to specifically kill 30 strains of <i>C. difficile</I>. We will aim to isolate the gene and fuse it to <i>Hcp</i> and <i>VgrG</i> genes which have been cloned without a stop codon. This endolysin interferes with the peptidoglycan layer of bacteria and causes the cell to lyse. By fusing this protein to proteins known to be exported during T6SS expression it should be able to target and kill <i>C. difficile</I>. <br><br> |
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- | <a href="https://static.igem.org/mediawiki/2012/ | + | <a href=" |
+ | https://static.igem.org/mediawiki/2012/8/84/Fusions1.jpg"><img src="https://static.igem.org/mediawiki/2012/b/bb/Fusions2.jpg"></a><br></center><br> | ||
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<p>Type IV secretion is thought to be mainly associated with exchange of genetic material between cells. In addition this system can target eukaryotic cells so was ruled out.</p> | <p>Type IV secretion is thought to be mainly associated with exchange of genetic material between cells. In addition this system can target eukaryotic cells so was ruled out.</p> | ||
- | <p>Type VI secretion systems were discovered in 2007 in <I>Vibrio cholera</I> and <I>Pseudomonas aeruginosa</I>. In these organisms, | + | <p>Type VI secretion systems were discovered in 2007 in <I>Vibrio cholera</I> and <I>Pseudomonas aeruginosa</I>. In these organisms, Type VI secretion is essential for their disease-causing ability. To date, T6SS genes have been shown to be present in the genomes of most Proteobacteria. Most of the original research on T6SS focused on its role in pathogenesis of higher organisms. However, more recent work tends to focus on the broad role T6SS plays in inter-bacterial interactions. In <i>Salmonella typhimurium</i>, the T6SS is encoded by 13 genes. Two of the proteins in this system, Hcp and VgrG, are known to be universally secreted and so as a result, these proteins will be fused with the endolysin. The other gene products are membrane proteins and thought to be involved in the assembly and function of the system.</p> |
- | <h3>Inflammation Biosensor</h3> | + | <h3>Inflammation Biosensor</h3><br> |
<center> | <center> | ||
- | <a href="https://static.igem.org/mediawiki/2012/d/de/Biosensor3.png"><img src="https://static.igem.org/mediawiki/2012/1/10/Biosensor2.png"></a><br></center | + | <a href="https://static.igem.org/mediawiki/2012/d/de/Biosensor3.png"><img src="https://static.igem.org/mediawiki/2012/1/10/Biosensor2.png"></a><br></center><br> |
- | <p>Although <i>E. coli</i> is a natural member of the gut flora, the presence of a T6SS may lead to increased competition with other organisms in the colon. In addition, there is a possibility that it could damage the cells of the gut epithelium. In order for the synthetic | + | <p>Although <i>E. coli</i> is a natural member of the gut flora, the presence of a T6SS may lead to increased competition with other organisms in the colon. In addition, there is a possibility that it could damage the cells of the gut epithelium. In order for the synthetic T6SS to only be expressed when required, a biosensor was incorporated into the project. The tetrathionate biosensor from <i>S. typhimurium</i> was chosen as the most appropriate for use in a synthetic bacterium that would eventually be intended for use in the gut. <i>C. difficile</i> induced pseudomembranous colitis of the gut causes the tissue to become inflamed. In the gut, thiosulphate is naturally produced from the conversion of Hydrogen suphide (released by the natural flora), in order to protect the tissue from this chemical. In the inflamed gut, reactive oxygen species are also frequently produced and these can convert the thiosulphate to tetrathionate. It is known that <i>S. typhimurium</i> is able to use tetrathionate as a terminal respiratory electron acceptor, with tetrathionate respiration carried out by the protein products of the ttrRSBCA genes. (Winters et. al. 2010)</br> |
- | For the biosensor produced in this project, two of the genes from | + | For the biosensor produced in this project, two of the genes from the <i>S. typhimurium</i> system were used: Ttr-R and Ttr-S. Ttr-S encodes for a membrane-bound histidine kinase sensor protein, whilst Ttr-R is the gene for a cytoplasmic DNA-binding response regulator. These act as a two component system which can turn on transcription of our T6SS genes in response to the presence of tetrathionate.</p> |
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- | <img src="https://static.igem.org/mediawiki/2012/ | + | <img src="https://static.igem.org/mediawiki/2012/0/04/Sponsorbar2.jpg"> |
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Latest revision as of 21:06, 26 September 2012
Continuing with the idea of reintroducing the natural gut bacteria to the colon in order to combat Clostridium difficile (C. difficile), our project aims to engineer a harmless Escherichia coli (E. coli) strain to target and kill C. difficile by expressing a specific endolysin. To do this, we will take a type VI secretion system (T6SS) from Salmonella typhimurium LT2 and express it in E. coli. In addition, we will take the C. difficile -specific endolysin gene from a bacteriophage and fuse it with genes from the T6SS that are universally secreted. In order to control the system, we will incorporate a synthetic inflammation biosensor which will ensure that the system is only activated during times of infection.
![](https://static.igem.org/mediawiki/2012/5/55/Solutionpic3.jpg)
© Eric Cascales, reproduced with kind permission
The Endolysin
The endolysin we have chosen to use is from ΦCD27. This has been shown by Mayer et. al. to specifically kill 30 strains of C. difficile. We will aim to isolate the gene and fuse it to Hcp and VgrG genes which have been cloned without a stop codon. This endolysin interferes with the peptidoglycan layer of bacteria and causes the cell to lyse. By fusing this protein to proteins known to be exported during T6SS expression it should be able to target and kill C. difficile.![](https://static.igem.org/mediawiki/2012/b/bb/Fusions2.jpg)
Secretion systems: Why type VI?
Type I secretion is a simple 3 protein system and was ruled out because it does not allow targeted secretion of a substrate.
Type II and type V secretion systems were also ruled out on the premise that they are unable to specifically secrete substrates to a target organism.
Type III secretion is a key system used by bacteria to deliver effector proteins to eukaryotic cells during pathogenesis. This was ruled out as we want to target C. difficile cells exclusively.
Type IV secretion is thought to be mainly associated with exchange of genetic material between cells. In addition this system can target eukaryotic cells so was ruled out.
Type VI secretion systems were discovered in 2007 in Vibrio cholera and Pseudomonas aeruginosa. In these organisms, Type VI secretion is essential for their disease-causing ability. To date, T6SS genes have been shown to be present in the genomes of most Proteobacteria. Most of the original research on T6SS focused on its role in pathogenesis of higher organisms. However, more recent work tends to focus on the broad role T6SS plays in inter-bacterial interactions. In Salmonella typhimurium, the T6SS is encoded by 13 genes. Two of the proteins in this system, Hcp and VgrG, are known to be universally secreted and so as a result, these proteins will be fused with the endolysin. The other gene products are membrane proteins and thought to be involved in the assembly and function of the system.
Inflammation Biosensor
![](https://static.igem.org/mediawiki/2012/1/10/Biosensor2.png)
Although E. coli is a natural member of the gut flora, the presence of a T6SS may lead to increased competition with other organisms in the colon. In addition, there is a possibility that it could damage the cells of the gut epithelium. In order for the synthetic T6SS to only be expressed when required, a biosensor was incorporated into the project. The tetrathionate biosensor from S. typhimurium was chosen as the most appropriate for use in a synthetic bacterium that would eventually be intended for use in the gut. C. difficile induced pseudomembranous colitis of the gut causes the tissue to become inflamed. In the gut, thiosulphate is naturally produced from the conversion of Hydrogen suphide (released by the natural flora), in order to protect the tissue from this chemical. In the inflamed gut, reactive oxygen species are also frequently produced and these can convert the thiosulphate to tetrathionate. It is known that S. typhimurium is able to use tetrathionate as a terminal respiratory electron acceptor, with tetrathionate respiration carried out by the protein products of the ttrRSBCA genes. (Winters et. al. 2010) For the biosensor produced in this project, two of the genes from the S. typhimurium system were used: Ttr-R and Ttr-S. Ttr-S encodes for a membrane-bound histidine kinase sensor protein, whilst Ttr-R is the gene for a cytoplasmic DNA-binding response regulator. These act as a two component system which can turn on transcription of our T6SS genes in response to the presence of tetrathionate.