Team:Dundee/Project

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             <p class="lead">What we did</p>
             <p class="lead">What we did</p>
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            <li class="list-group-item"><a href="#0" class="">Initial planning and cloning strategy</a>
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            <li class="list-group-item"><a href="#1" class="">Building the PQS sensor</a>
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      <h1>The <i>Pseudomonas</i> Quinolone Signal (PQS) sensing system: What it is and how it works</h1>
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<i>Pseudomonas</i>quinolone signal (2-heptyl-3-hydroxy-4-quinolone) is a quorum-sensing molecule produced by <i>Pseudomonas aeruginosa</i>, which regulates the expression of genes involved in biofilm development and virulence.<sup>1</sup> Expression of these traits is mediated through the LysR-type transcriptional regulator, PqsR. Sequence analysis of PqsR predicts that it is a soluble protein but fractionation of <i>P. aeruginosa</i> has shown that the protein is primarily associated with the inner membrane.<sup> 2</sup> It is not clear whether this is through the interaction with membrane lipids or with an unidentified integral inner membrane protein. In the presence of PQS, PqsR interacts with the promoter region of the <i>pqsABCDE</i> operon, allowing transcription of the downstream genes.<sup>3</sup> We have engineered<i> E. coli</i> to express this signal transduction system for the detection of PQS, with a promoter-less mCherry fused to the <i>pqsA</i> promoter to give a fluorescent output.
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Chromosomal DNA from <i>Pseudomonas Aeruginosa</i> PA01 strain was kindly gifted to us by Robert Ryan and Shi-Qi An from The Division of Molecular Microbiology in the College of Life sciences at the University of Dundee. This was used as a template for the amplification of the <i>pqsA</i> promoter.
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The <i>pqsA</i> promoter region was cloned into the pSB1C3 plasmid (to give Biobrick (BBa_K1315001), and was then subcloned into pBluescript. Promoterless mCherry was amplified using BBa K562011 as a template and was cloned into pBluescript downstream of the <i>pqsABCDE</i> promoter. The P<i>pqsA</i>-mCherry construct was then subcloned into pUniprom.
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The pqsR gene (present in BBa_K1157001) which was designed and cloned by Lin Ang Chieh from iGEM13-NTU-Taida was used as a template for the amplification of pqsR with an
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influenza virus haemagglutinin (HA) tag coding sequence, which can be detected with commercial antibodies. This tag was added to the C-terminus of the protein to facilitate immunohistochemistry.
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The plasmid was verified by sequencing.
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The completed construct was transformed into <i>E. coli </i>strain MC1061 as a chassis for our biosensor.
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Initially, western blots were undertaken to test for the production of PqsR-HA. We transformed our plasmid encoding PqsR-HA into <i>E. coli </i>strain MC1061 and blotted for recombinant protein production. An overnight culture of the cells was lysed and proteins separated by SDS-PAGE in a 12% acrylamide gel. Anti-HA antibodies linked to horseradish peroxidase were used for detection of PqsR. Fig 3A shows the expression of PqsR within our system.
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As shown in Fig 3, successful production of PqsR-HA (expected mass 38 kDa) was observed. With the PqsR regulator being produced we could begin to test for a response to PQS. To test how the system would respond to PQS, cells containing the construct were cultured in LB medium and spiked with synthetic PQS in DMSO at concentrations of 50μM and 500μM. A western blot with anti-mCherry antibodies was performed on the treated cells alongside an un-spiked, PQS-negative control. Two further controls - MC1061 cells harbouring the empty pUniprom vector (mCherry negative control) or BBa K562011 (mCherry positive control) were also included.
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As shown in Fig 4 incubation of cells with synthetic PQS, did not induce expression of mCherry. It is not clear why the biosensor did not respond. One possibility we considered is that the PqsR is not correctly localized to the membrane in the <i>E. coli</i> chassis. We therefore fractionated <i>E. coli</i> cells containing PqsR-HA into soluble and membrane fractions. However, as shown in Fig 5, all of the detectable PqsR-HA was found in the membranes as it is in the native organism.
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We discussed the failure of the PQS biosensor to respond extensively with the modelling team (include link). The feedback we received was that increasing the number of promoters (by at least 10-fold) should allow for sufficient detection of mCherry. We are in the process of building this new expression system. </p>
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            <td>PqsA</td>
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<!-- END OF PQS-->
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<h1>The <i>Burkholderia</i> Diffusible signalling Factor (BDSF) sensing system: What it is and how it works</h1>
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Cis-2 fatty acids are used by many bacterial species as signalling molecules to facilitate inter- and intra-species communication and as a method of regulation of gene expression. <i>Burkholderia</i> Diffusible Signalling Factor (BDSF) is a Cis-2 dodecenoic acid that is produced exclusively by the pathogenic bacteria of the <i>Burkholderia cepacia </i>complex where it regulates expression of genes involved in virulence<sup>1,2</sup>. It is structurally similar to, but distinct from, DSF (cis-11-methyl-2-dodecenoic acid) produced by <i>Stenotrophomonas maltophilia</i>.
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In <i>B. cenocepacia</i> BDSF activates gene expression through a two component phosphorelay. BCAM0227 is a transmembrane histidine kinase which phosphorylates the response regulator BCAM0228 in the presence of exogenous BDSF. BCAM0228 then binds and activates transcription of <i>cblD</i>, a gene involved in <i>Burkholderia virulence</i><sup>2</sup>.
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We engineered <i>E. coli</i> to express this signal transduction system for the detection of BDSF, with a promoter-less <i>gfp</i> gene downstream of the <i>cblD</i> promoter. With this, our sensor will detect any BDSF in its environment via the BCAM0227 receptor and activate GFP production.
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Chromosomal DNA from <i>Burkholderia cenocepacia</i> J2315 was kindly gifted to us by Drs Robert Ryan and Shi-Qi An from the Division of Molecular Microbiology in the College of Life Sciences at the University of Dundee. This was used as template for the amplification of BCAM0228 and the cblD promoter region.
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The <i>cblD</i> promoter region was cloned into the pSB1C3 plasmid (to give Biobrick BBa_K1315008), and was then subcloned into pBluescript. Promoterless <i>gfp</i> was amplified using BBa_K562012 as a template, and was cloned into pBluescript downstream of the <i>cblD</i> promoter. The <i>manA</i> promoter-<i>gfp</i> construct was then subcloned into pUniprom.
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A modified version of the BCAM0227 gene which was compatible with biobrick specifications and standards was synthesised by a third party (Dundee Cell Products). This was subsequently subcloned into the pUniprom vector harbouring P<sub>cblD</sub>-<i>gfp</i>.
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To adhere to the iGEM rules and regulations, it was necessary to remove an illegal <i>Eco</i>RI restriction site present in <i>BCAM0228</i>. The modified gene was cloned into the pSB1C3
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plasmid (to give Biobrick BBa_K1315007). <i>BCAM0228</i> was then subcloned into the pUniprom vector that already harboured P<sub>manA</sub>-<i>gfp</i> and <i>BCAM0227</i>. To facilitate immunochemistry we chose to supply BCAM0227 and BCAM0228 with an influenza virus hemagglutinin (HA) tag which can be detected with commercial antibodies. This tag was added to the C-terminus of each protein.
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The plasmid was verified by sequencing.
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The completed construct was transformed into MC1061 <i>E. coli</i> as a chassis for our biosensor.
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Initially, Western blots were undertaken to test for the sequential production of BCAM0227-HA and BCAM0228-HA.  An overnight culture of the cells were lysed and proteins separated by SDS-PAGE in a 12% acrylamide gel. Anti-HA antibodies linked to horseradish peroxidase were used for detection of BCAM0227 and BCAM0228. Fig 3 shows that both of the proteins are being expressed in the system.
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With all of the components of the system being produced, we could begin to test for a response to BDSF. To test how the system would respond to BDSF, cells containing the construct were cultured in LB medium and spiked with synthetic BDSF in DMSO at concentrations of 50µM, which corresponds to the levels found in the sputum of lungs colonised by <i>Burkholderia</i><sup>3</sup> and 500µM. A western blot with anti-GFP antibodies was performed on the treated cells alongside an un-spiked, BDSF-negative control, and MC1061 cells harbouring the empty pUniprom vector. The results are shown in Fig 4.
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These results indicate that GFP production is activated regardless of the presence of BDSF. To test whether our <i>E. coli</i> chassis was responsible for activating GFP production, either via phosphorylation of BCAM0228 or by directly activating the <i>cblD</i> promoter, we made a new construct harbouring <i>cblD-gfp</i> and <i>BCAM0228-HA</i>, but lacking <i>BCAM0227</i>. Fig 5 shows that GFP was only produced in the presence of BCAM0228-HA. We therefore concluded that GFP output is likely caused by crosstalk by two component regulatory systems within <i>E. coli</i> promoting the phosphorylated state of the BCAM0228 response regulator.
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            <img  data-alt-src="https://static.igem.org/mediawiki/2014/2/2c/BCAM228_GFP.png" src="https://static.igem.org/mediawiki/2014/2/2c/BCAM228_GFP.png" />                             
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    </a>
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            <p>
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We are continuing to investigate this issue by screening <i>E. coli</i> mutants carrying deletions in genes coding for sensor kinases, taking advantage of the <i>E. coli</i> Keio mutant collection<sup>3</sup>. Alternatively it may be that BCAM0228 is being phosphorylated by a small molecular weight phosphate donor such acetylphosphate. Acetylphosphate production can be eliminated in a <i>pta-ackA</i> double deletion mutant. Further work is required before the BDSF sensor is fully functional.
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</br>
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The Following parts were deposited as Biobricks
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<table class="table">
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            <th>Part</th>
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            <th>Registry</th>
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    <tbody>
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            <td>BCAM0228</td>
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            <td>BDSF receptor/histidine kinase</td>
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            <td>BBa_K1315007</td>
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        </tr>
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        <tr>
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            <td>CblD</td>
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            <td>BCAM0228 inducible promoter</td>
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            <td>BBa_K1315008</td>
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        </tr>
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    </tbody>
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</table>
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</p>
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<!-- END OF BDSF -->
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<h1>The Diffusible signalling Factor (DSF) sensing system: What it is and how it works</h1>
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            <h2 id="0">Initial planning and cloning strategy</h2>
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      <a href="#" class= "system pull-left">
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            <img src="https://static.igem.org/mediawiki/2014/e/ea/DSF_system.png" />                             
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    </a> 
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            <p>
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Diffusible signalling Factor (DSF) is a long chain cis-unsaturated fatty acid (link to Jess background?) produced by bacteria of the Xanthomonas genus, including the CF lung pathogen Stenotrophomonas maltophilia 1,2.  To build our synthetic DSF detector, we chose to reconstitute the well-characterised DSF signal perception system from Xanthomonas campestris in our E. coli chassis. As can be seen in Fig 1, the RpfC/RpfG system, which perceives and responds to DSF, is more complex than the average two component system. DSF is detected at the periplasmic side of the membrane by the RpfC sensor protein, which autophosphorylates and then transfers its phospho group to the  cytoplasmic response regulator RpfG. However, instead of acting as a direct transcriptional regulator phosphorylated RpfG acts as a phosphodiesterase cleaving the second-messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) into 2 GMP molecules3,4. This reduction in cellular c-di-GMP relieves inhibition of the transcriptional activator Clp, which is then free to bind to the manA promoter and allow transcription of the downstream genes5. In our synthetic system we fused the manA promoter to promoter-less GFP (GFP biobrick hyperlink in here) to give a fluorescent output.
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</p>
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            <hr>
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            <h2 id="1">Building the PQS sensor</h2>
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          <div class="row">
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<div class="col-xs-12">
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            <p>
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Chromosomal DNA from <i>Pseudomonas Aeruginosa</i> PA01 strain was kindly gifted to us by Robert Ryan and Shi-Qi An from The Division of Molecular Microbiology in the College of Life sciences at the University of Dundee. This was used as a template for the amplification of the <i>pqsA</i> promoter.
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The <i>pqsA</i> promoter region was cloned into the pSB1C3 plasmid (to give Biobrick (BBa_K1315001), and was then subcloned into pBluescript. Promoterless mCherry was amplified using BBa K562011 as a template and was cloned into pBluescript downstream of the <i>pqsABCDE</i> promoter. The P<i>pqsA</i>-mCherry construct was then subcloned into pUniprom.
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The pqsR gene (present in BBa_K1157001) which was designed and cloned by Lin Ang Chieh from iGEM13-NTU-Taida was used as a template for the amplification of pqsR with an
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influenza virus haemagglutinin (HA) tag coding sequence, which can be detected with commercial antibodies. This tag was added to the C-terminus of the protein to facilitate immunohistochemistry.
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    <a href="#" class= "system pull-left">
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            <img src="https://static.igem.org/mediawiki/2014/1/1f/DSF_plasmid.png" />                             
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            <p>
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The plasmid was verified by sequencing.
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            <hr>
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            <h2 id="2">Characterisation</h2>
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            <p>
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A series of western blots were performed to test for the sequential production of each of the HA-tagged proteins. We transformed our plasmid series (encoding RpfC-HA, RpfC-HA + Clp-HA and RpfC-HA +Clp-HA + RpfG-HA) into E. coli strain MC1061 and blotted for recombinant protein production.
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</p>
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    <a href="#" class= "system pull-left">
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            <img src="https://static.igem.org/mediawiki/2014/e/ea/RpfC-Clp_HA.png" />                             
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    </a> 
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            <p>
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As shown in Fig 3, successful production of RpfC-HA (expected mass 81kDa), RpfG-HA (expected mass of 43kDa) and Clp-HA (expected mass 27kDa) was observed. We then went on to test our DSF detector by incubating E. coli producing the three HA-tagged proteins with increasing concentrations of pure DSF (obtained from Sigma) and monitoring for production of GFP from the manA promoter by Western blotting. As shown in Fig 4A, we were able to specifically detect GFP production in E. coli cells harbouring our DSF-detection plasmid. However we noted that GFP was being produced in the absence of exogenous DSF addition. We reasoned that this could be due to either: (i) the endogenous activation of the manA by an E. coli transcriptional regulator or (ii) the presence of DSF or a DSF-like compound in the cells and/or growth medium. To distinguish between these two possibilities we introduced pUniprom carrying the manA-gfp fusion alone (i.e. in the absence of the rpfC, rpfG and clp genes) into E. coli and blotted for GFP production. As shown in Fig 4B, GFP was produced by E. coli in the absence of the X. campestris DSF sensing proteins. We therefore conclude that the manA promoter is recognised by an E. coli transcription factor.
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    <a href="#" class= "system pull-left">
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            <img src="https://static.igem.org/mediawiki/2014/d/d0/Rpfc_clp_rpfg_GFP.png" />                             
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    </a> 
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</div>
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<div class="col-xs-12">
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<p>
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For the DSF sensing system to work we need to eliminate the background activation of PmanA-gfp. Since the X. campestris Clp protein is very similar to the E. coli cyclic AMP receptor protein (CRP) transcripton factor (46% identity, 71% similarity over the whole length of the protein), we next tested whether the production of GFP was dependent upon CRP. To this end we used a crp deletion strain from the E. coli Keio mutant collection6 and compared GFP production in this strain background with the cognate wild type strain (Fig 5). However GFP was still produced (although possibly at a lower level) than in the crp+ background.
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</p>
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    <a href="#" class= "system pull-left">
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            <img src="https://static.igem.org/mediawiki/2014/3/33/Delta_CRP_GFP.png" />                             
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    </a> 
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</div>
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</div>
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<div class="row">
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<div class="col-xs-12">
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<p>
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We are continuing to investigate this issue by screening other E. coli mutants carrying deletions in genes coding for transcription factors which show similarity to Clp, for example the anaerobic regulator, FNR. We anticipate that it may be necessary to make combinatorial mutations to completely eliminate exogenous GFP production.
+
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</p>
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</div>
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</div>
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+
-
<table class="table">
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    <thead>
+
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        <tr>
+
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            <th>Part</th>
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-
            <th>Description</th>
+
-
            <th>Registry</th>
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-
        </tr>
+
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    </thead>
+
-
    <tbody>
+
-
        <tr>
+
-
            <td>rpfC</td>
+
-
            <td>DSF Sensor Kinase</td>
+
-
            <td>BBa_K1315002</td>
+
-
        </tr>
+
-
        <tr>
+
-
            <td>rpfG</td>
+
-
            <td>Two-component system regulator activated by sensor kinase</td>
+
-
            <td>BBa_K1315003</td>
+
-
        </tr>
+
-
        <tr>
+
-
            <td>ydeH</td>
+
-
            <td>Zinc-sensory diguanylate cyclase</td>
+
-
            <td>BBa_K1315004</td>
+
-
        </tr>
+
-
        <tr>
+
-
            <td>Clp</td>
+
-
            <td>Clp Transcriptional activator inhibited by c-di-GMP</td>
+
-
            <td>BBa_K1315005</td>
+
-
        </tr>
+
-
        <tr>
+
-
            <td>manA</td>
+
-
            <td>manA gene promoter activated by Clp</td>
+
-
            <td>BBa_K1315006</td>
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-
        </tr>
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-
    </tbody>
+
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</table>
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Latest revision as of 21:05, 9 October 2014

Dundee 2014

Dundee 2014

Project

What we did

References

Retrieved from "http://2014.igem.org/Team:Dundee/Project"