Team:University of Sheffield /Project

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{| style="color:#ffffff;background-color:#6633ff;" cellpadding="5" cellspacing="2" border="2" bordercolor="blue" width="85%" align="center"
!align="center"|[[Team:University_of_Sheffield |Home]]
!align="center"|[[Team:University_of_Sheffield |Home]]
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!align="center"|[[Team:University_of_Sheffield / Team |The Team]]
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!align="center"|[[Team:University_of_Sheffield /Project|Our project]]
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!align="center"|[[Team:University_of_Sheffield /Project|The Project]]
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!align="center"|[[Team:University_of_Sheffield /Modelling|Modelling]]
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!align="center"|[[Team:University_of_Sheffield /Lab Books| Lab Books]]
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!align="center"|[[Team:University_of_Sheffield /Administration|Administration]]
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|<H2>Project Overview</H2><br>
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<p>We want is to engineer a biological machine that will sense microbial contamination of drinking water using quorum sensing, namely the system in '''''Vibrio cholerae''''', the bacterial species that causes epidemics of cholera. Quorum sensing systems use sensing proteins in the membrane (<em>[http://en.wikipedia.org/wiki/Histidine_kinase histidine-kinases] in this case</em>) to detect molecules excreted by the bacteria’s own species. After detection in the membrane, a phosphate signal is passed down pathway, causing a DNA regulatory effect. Each quorum-sensing species also has its own corresponding quorum-producing protein. In Vibrio cholerae the sensing protein is CqsS and the quorum-producing protein CqsA.</p>
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<font size="7" color="#151B8D" face="TUOS Stephenson"><U>'''Overall project'''</U>
 
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<font size="5" color="#151B8D" face="TUOS Blake"><u>'''Project Overview'''</u>
 
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The aim is to engineer a biological machine that will sense microbial contamination of drinking water using quorum sensing, namely, '''''Vibrio cholerae''''', the bacterial species that causes epidemics of cholera. Quorum sensing systems use sensing proteins in the membrane (<em>[http://en.wikipedia.org/wiki/Histidine_kinase histidine-kinases] in this case</em>) to detect molecules excreted by the bacteria’s own species. After detection in the membrane, a phosphate signal is passed down pathway, causing a DNA regulatory effect. Each quorum-sensing species also has its own corresponding quorum-producing protein. In Vibrio cholerae the sensing protein is CqsS and the quorum-producing protein CqsA.
 
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<font size="5" color="#151B8D" face="TUOS Blake"><u>'''If everything goes well...'''</u>
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<p>We are hijacking a pathway already present in E.coli – the non-essential BarA, pathway which is partly responsible for 'last resort' carbon metabolism. This pathway starts with similar histidine-kinase (<em>KdpD</em>) to Vibrio cholerae, and makes a compatible candidate topologically. We're taking the sending part of the Vibrio cholerae histidine kinase (called CqsS) and attaching it to the phosphotransfer part of the BarA histidine kinase, forming a new, fusion kinase. This is the simple way of putting it, as fusion proteins are notoriously difficult to get to work in reality. However the similiar topology between BarA and CqsS helps us somewhat, and sessions with Prof Rice (a protein crystollogropher in the Molecular Biology and Biotechnology department here) have increased our chances of a succesfulm protein.
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When triggered, out fusion kinase will pass the signal down E.coli's natural BarA response system to its target genes, with which we are fusing GFP. In short, is cholera autoinducers are in the water, our cell will glow! The original BarA will be knocked out of E.coli to make way for recombinant receptors (we dont want native BarA triggering our GFP!).
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Our organism would initially sense Vibrio cholerae. This will be done by hijacking a pathway already present in E.coli – the non-essential kdp pathway which is partly responsible for K+ detection and response regulation. This pathway has a similar histidine-kinase as its sensing molecule (<em>KdpD</em>) to Vibrio cholerae and Legionella, and results in a strong DNA response. The original KdpD will be knocked out of E.coli to make way for recombinant receptors.
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The recombinant receptor will be made from the sensing part of the pathogens quorum sensing protein, fused with the signaling part of KdpD. Due to the similarity of the two receptors in certain regions, this should allow quorums to be detected using the pathogens sensing mechanism, but the signal to be passed to the kdp pathway. The target genes at the end of the kdp pathway will be modified so as to express the bioluminescent, luciferase, as an indicator there are quorums (<em>and therefore pathogens</em>)present. [http://en.wikipedia.org/wiki/Luciferase Luciferase] was chosen due to its ability to be detected without expensive equipment, and the possibility of targeting proteases for its degradation if we chose to make the machine re-useable.     
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The recombinant protein will be expressed on a high-copy plasmid, so we should be able to get large-scale production of the recombinant proteins which may lower the threshold number of quorum molecules required to trigger the pathway.</p>
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Two recombinant proteins will be produced, one made from CqsS and KdpD, and the other LqsS and KdpD, each destined for different E.coli. Using a high-copy plasmid we should be able to get large-scale production of the recombinant proteins which may lower the threshold number of quorum molecules required to trigger the pathway. The result is two E.coli strains, capable of sensing either Vibrio cholerae or Legionella.
 
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<font size="5" color="#151B8D" face="TUOS Blake"><u>'''History of Development'''</u>
<font size="5" color="#151B8D" face="TUOS Blake"><u>'''History of Development'''</u>

Revision as of 18:09, 27 September 2008


Home Our project Modelling Parts Lab Books Calendar Administration

Sheffield igem logo.JPG

Project Overview


We want is to engineer a biological machine that will sense microbial contamination of drinking water using quorum sensing, namely the system in Vibrio cholerae, the bacterial species that causes epidemics of cholera. Quorum sensing systems use sensing proteins in the membrane ([http://en.wikipedia.org/wiki/Histidine_kinase histidine-kinases] in this case) to detect molecules excreted by the bacteria’s own species. After detection in the membrane, a phosphate signal is passed down pathway, causing a DNA regulatory effect. Each quorum-sensing species also has its own corresponding quorum-producing protein. In Vibrio cholerae the sensing protein is CqsS and the quorum-producing protein CqsA.


If everything goes well...


We are hijacking a pathway already present in E.coli – the non-essential BarA, pathway which is partly responsible for 'last resort' carbon metabolism. This pathway starts with similar histidine-kinase (KdpD) to Vibrio cholerae, and makes a compatible candidate topologically. We're taking the sending part of the Vibrio cholerae histidine kinase (called CqsS) and attaching it to the phosphotransfer part of the BarA histidine kinase, forming a new, fusion kinase. This is the simple way of putting it, as fusion proteins are notoriously difficult to get to work in reality. However the similiar topology between BarA and CqsS helps us somewhat, and sessions with Prof Rice (a protein crystollogropher in the Molecular Biology and Biotechnology department here) have increased our chances of a succesfulm protein. When triggered, out fusion kinase will pass the signal down E.coli's natural BarA response system to its target genes, with which we are fusing GFP. In short, is cholera autoinducers are in the water, our cell will glow! The original BarA will be knocked out of E.coli to make way for recombinant receptors (we dont want native BarA triggering our GFP!). The recombinant protein will be expressed on a high-copy plasmid, so we should be able to get large-scale production of the recombinant proteins which may lower the threshold number of quorum molecules required to trigger the pathway.


History of Development

More Project Details

Parts submitted to the Registry
Modelling

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