Team:Dundee/Project/PAI-1

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              <h1>Pseudomonas auto inducer system
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<h1><font size="14">The <i>Pseudomonas</i> Autoinducer-1 (PAI-1) Sensing System</font></h1>
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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="#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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             <li class="list-group-item"><a href="#1" class="">Building the PAI-1 Sensor</a>
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<i>Pseudomonas</i> autoinducer-1 (N-3-oxododecanoyl homoserine lactone) is a second quorum sensing molecule produced by <i>Pseudomonas aeruginosa</i> that works in concert with LasR to increase the expression of a number of virulence genes, including those for several proteases (<i>lasB</i>, <i>lasA</i>, <i>aprA</i>) and exotoxin A (<i>toxA</i>)<sup>1</sup>.
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<a href="http://parts.igem.org/Part:BBa_C0179">LasR</a> is a transcriptional activator that binds the autoinducer molecule PAI-1, causing the protein to dimerise and to activate transcription of various promoters including that of the <a href="http://parts.igem.org/Part:BBa_R0079"><i>lasB</i> </a>gene<sup>2</sup>. It has been reported that the LasR-PAI complex can also activate the <i>Vibrio fischeri </i> <a href="http://parts.igem.org/Part:BBa_R0062"><i>luxR</i> </a>promoter<sup>2,3</sup>. Both P<i><sub>lasB</sub></i> and P<i><sub>luxe</sub></i> were adopted as LasR-PAI-1 inducible promoters in our device. Using pre-existing BioBricks we have designed new circuits to engineer <i>E. coli</i> to express the LasR transduction system for the detection of PAI-1 (as shown in Fig 1), along with promoter-less <a href="http://parts.igem.org/Part:BBa_E0040"><i>gfp</i></a> fused to either the <i>lasB</i> or <i>luxR</i> promoter.
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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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            <h2 id="1">Building the PAI-1 sensor</h2>
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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 <a href="http://parts.igem.org/Part:BBa_K1315001">BBa_K1315001</a>), 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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All the parts we used to construct our PAI-1 sensor were obtained from the iGEM parts registry (detailed below in Fig 2), and sequentially cloned into pSB1C3 plasmid. The gene encoding the green fluorescent protein (GFP) was fused to our sensing device.  
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The pqsR gene (present in <a href="http://parts.igem.org/Part:BBa_K1315001">BBa_K1315001</a>) which was designed and cloned by Lin Ang Chieh from iGEM13-NTU-Taida was used as a template for the amplification of <i>pqsR</i> 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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            <th>Part</th>
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            <th>Description</th>
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            <th>Registry</th>
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            <td>P<i><sub>tet</sub></i></td>
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            <td>TetR repressible promoter</td>
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            <td><a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a></td>
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            <td>LasR CDS</td>
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            <td>LasR activator from <i>P. aeruginosa</i> PAO1</td>
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            <td><a href="http://parts.igem.org/Part:BBa_C0179">BBa_C0179 </a></td>
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            <td>LasR and PAI regulated promoter </td>
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            <td>Binding region for LasR protein</td>
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            <td><a href="http://parts.igem.org/Part:BBa_R0079">BBa_R0079</a></td>
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            <td>P<i><sub>luxR</sub></i></td>
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            <td>Promoter</td>
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            <td><a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a></td>
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            <td><i>gfp</i> </td>
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            <td>green fluorescent protein</td>
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            <td><a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a></td>
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The plasmids were verified by sequencing.
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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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The completed construct was transformed into <i>E. coli</i> strain MC1061 as a chassis for our two PAI biosensors.  
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            <h2 id="2">Characterisation</h2>
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With all of the components of the system in place, we could begin to test for a response to PAI-1. To this end, cells containing the construct were cultured in LB medium, cultures were spiked with 500μM synthetic PAI-1 in DMSO and samples were withdrawn at time periods of up to one hour following PAI-1 addition. A western blot with anti-GFP antibodies was performed on the treated cells alongside an un-spiked, PAI-1 negative control, and MC1061 cells harbouring the empty pSB1C3 vector. The results are shown in Fig 3.  
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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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As shown in Fig 3A, GFP production is activated by PAI-1 following a 60 minute induction period. Thus the P<sub><i>lasB</sub></i>LasR circuit has responded as expected to the presence of PAI-1. Fig 3B, shows a high basal GFP production driven by P<i><sub>luxR</sub></i> in the absence of the autoinducer molecule (PAI-1), however there is increased GFP production over time in the presence of PAI-1. The basal GFP production seen from P<i><sub>luxR</sub></i> in the absence of PAI-1 may reflect the fact that this is a ‘foreign’ promoter from <i>V. fischeri</i> and is not naturally regulated by LasR. Therefore regulation from this hybrid system might expected to be non-optimal. None-the-less these data show that both of our engineered systems respond to PAI-1.
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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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The following parts were deposited as BioBricks:
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             <td>PAI-1 activated system 1</td>
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             <td><a href="http://parts.igem.org/Part:BBa_K1315009">BBa_K1315009</a></td>
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            <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>luxR</sub></i>-<i>gfp</i></td>
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            <td>PAI-1 activated system 2</td>
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            <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>lasB</sub></i></td>
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            <td>Intermediate part</td>
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            <td><a href="http://parts.igem.org/Part:BBa_K1315011">BBa_K1315011</a></td>
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            <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>luxR</sub></i></td>
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            <td>Intermediate part</td>
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            <td><a href="http://parts.igem.org/Part:BBa_K1315012">BBa_K1315012</a></td>
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            <h3>References</h3>
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<sup>1</sup>Pearson, J.P. et al. (1997). Journal of Bacteriology 18, 5756-5767<br>
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<sup>2</sup>Pearson, J.P (1994). Microbiology 92, 1490-1494<br>
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<sup>3</sup>Kievit T. et al (1999) J.Bacteriol 7, 2175-2184<br>
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{{:Team:Dundee/Template/welcomeNoteSchool}}

Latest revision as of 22:21, 17 October 2014

Dundee 2014

Dundee 2014

The Pseudomonas Autoinducer-1 (PAI-1) Sensing System

Initial Planning and Cloning Strategy


Pseudomonas autoinducer-1 (N-3-oxododecanoyl homoserine lactone) is a second quorum sensing molecule produced by Pseudomonas aeruginosa that works in concert with LasR to increase the expression of a number of virulence genes, including those for several proteases (lasB, lasA, aprA) and exotoxin A (toxA)1. LasR is a transcriptional activator that binds the autoinducer molecule PAI-1, causing the protein to dimerise and to activate transcription of various promoters including that of the lasB gene2. It has been reported that the LasR-PAI complex can also activate the Vibrio fischeri luxR promoter2,3. Both PlasB and Pluxe were adopted as LasR-PAI-1 inducible promoters in our device. Using pre-existing BioBricks we have designed new circuits to engineer E. coli to express the LasR transduction system for the detection of PAI-1 (as shown in Fig 1), along with promoter-less gfp fused to either the lasB or luxR promoter.

Building the PAI-1 sensor

All the parts we used to construct our PAI-1 sensor were obtained from the iGEM parts registry (detailed below in Fig 2), and sequentially cloned into pSB1C3 plasmid. The gene encoding the green fluorescent protein (GFP) was fused to our sensing device.





Part Description Registry
Ptet TetR repressible promoter BBa_R0040
LasR CDS LasR activator from P. aeruginosa PAO1 BBa_C0179
LasR and PAI regulated promoter Binding region for LasR protein BBa_R0079
PluxR Promoter BBa_R0062
gfp green fluorescent protein BBa_E0040

The plasmids were verified by sequencing.

The completed construct was transformed into E. coli strain MC1061 as a chassis for our two PAI biosensors.

Characterisation

With all of the components of the system in place, we could begin to test for a response to PAI-1. To this end, cells containing the construct were cultured in LB medium, cultures were spiked with 500μM synthetic PAI-1 in DMSO and samples were withdrawn at time periods of up to one hour following PAI-1 addition. A western blot with anti-GFP antibodies was performed on the treated cells alongside an un-spiked, PAI-1 negative control, and MC1061 cells harbouring the empty pSB1C3 vector. The results are shown in Fig 3.


As shown in Fig 3A, GFP production is activated by PAI-1 following a 60 minute induction period. Thus the PlasBLasR circuit has responded as expected to the presence of PAI-1. Fig 3B, shows a high basal GFP production driven by PluxR in the absence of the autoinducer molecule (PAI-1), however there is increased GFP production over time in the presence of PAI-1. The basal GFP production seen from PluxR in the absence of PAI-1 may reflect the fact that this is a ‘foreign’ promoter from V. fischeri and is not naturally regulated by LasR. Therefore regulation from this hybrid system might expected to be non-optimal. None-the-less these data show that both of our engineered systems respond to PAI-1.

The following parts were deposited as BioBricks:

Part Description Registry
Ptet-lasR-PlasB-gfp PAI-1 activated system 1 BBa_K1315009
Ptet-lasR-PluxR-gfp PAI-1 activated system 2 BBa_K1315010
Ptet-lasR-PlasB Intermediate part BBa_K1315011
Ptet-lasR-PluxR Intermediate part BBa_K1315012

References

1Pearson, J.P. et al. (1997). Journal of Bacteriology 18, 5756-5767
2Pearson, J.P (1994). Microbiology 92, 1490-1494
3Kievit T. et al (1999) J.Bacteriol 7, 2175-2184