Team:Dundee/Project/PAI-1

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              <h1>Pseudomonas auto inducer-1 sensing 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 PAI-1 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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             <li class="list-group-item"><a href="#2" class="">Characterisation</a>  
             <li class="list-group-item"><a href="#2" class="">Characterisation</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 (lasB, lasA, aprA) and exotoxin A (toxA)<sup>1</sup>.
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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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LasR is a transcriptional activator that binds the auto-inducer molecule PAI-1, causing the protein to dimerise and to activate transcription of various promoters including that of the <i>lasB</i> gene<sup>2</sup>.. It has been reported that the LasR-PAI complex can also activate the <i>Vibrio fischeri luxR</i> promoter<sup>2,3</sup>. Both <i>PlasB</i> and <i>PluxR</i> were adopted as LasB-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, along with promoter-less <i>gfp</i> fused to either the <i>lasB</i> or <i>luxR</i> promoter.
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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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             <h2 id="1">Building the PAI-1 sensor</h2>
             <h2 id="1">Building the PAI-1 sensor</h2>
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All the parts we used to construct our PAI-1 sensor were obtained from the iGEM parts registry and sequentially cloned into pSB1C3 plasmid. The gene encoding the green fluorescent protein (GFP) was fused to our sensing device.  
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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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             <td>pTet</td>
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             <td>P<i><sub>tet</sub></i></td>
             <td>TetR repressible promoter</td>
             <td>TetR repressible promoter</td>
             <td><a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a></td>
             <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 P.aeruginosa PAO1</td>
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             <td>LasR activator from <i>P. aeruginosa</i> PAO1</td>
             <td><a href="http://parts.igem.org/Part:BBa_C0179">BBa_C0179 </a></td>
             <td><a href="http://parts.igem.org/Part:BBa_C0179">BBa_C0179 </a></td>
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             <td>PluxR</td>
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             <td>P<i><sub>luxR</sub></i></td>
             <td>Promoter</td>
             <td>Promoter</td>
             <td><a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a></td>
             <td><a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a></td>
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             <td>GFP </td>
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             <td><i>gfp</i> </td>
             <td>green fluorescent protein</td>
             <td>green fluorescent protein</td>
             <td><a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a></td>
             <td><a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a></td>
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The plasmids were verified by sequencing.
The plasmids were verified by sequencing.
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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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The completed construct was transformed into E. coli 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.  
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 3A, GFP production is activated by PAI-1 following a 60 minute induction period. Thus the LasR/plasB 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 auto-inducer molecule (PAI-1), however there is increased GFP production over time in the presence of PAI-1. The basal GFP production seen from <i>PluxR</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 LasB. 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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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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The following parts were deposited as Biobricks
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The following parts were deposited as BioBricks:
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             <td>Ptet-lasR-plasB-GFP</td>
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             <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>lasB</sub></i>-<i>gfp</i></td>
             <td>PAI-1 activated system 1</td>
             <td>PAI-1 activated system 1</td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315009">BBa_K1315009</a></td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315009">BBa_K1315009</a></td>
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             <td>Ptet-lasR-pluxR-GFP</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>
             <td>PAI-1 activated system 2</td>
             <td>PAI-1 activated system 2</td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315010">BBa_K1315010</a></td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315010">BBa_K1315010</a></td>
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             <td>Ptet-lasR-plasB</td>
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             <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>lasB</sub></i></td>
             <td>Intermediate part</td>
             <td>Intermediate part</td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315011">BBa_K1315011</a></td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315011">BBa_K1315011</a></td>
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             <td>Ptet-lasR-pluxR</td>
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             <td>P<i><sub>tet</sub></i>-<i>lasR</i>-P<i><sub>luxR</sub></i></td>
             <td>Intermediate part</td>
             <td>Intermediate part</td>
             <td><a href="http://parts.igem.org/Part:BBa_K1315012">BBa_K1315012</a></td>
             <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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1 Pearson, J.P. et al. (1997). Journal of Bacteriology 18, 5756-5767<br>
 
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3 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