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Team:Dundee/Project/PAI-1
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| + | <h1><font size="14">The <i>Pseudomonas</i> Autoinducer-1 (PAI-1) Sensing System</font></h1> | ||
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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>. | <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>. | ||
| - | <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>lasB</i> and P<i> | + | <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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<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> | <td>LasR CDS</td> | ||
| - | <td>LasR activator from P.aeruginosa PAO1</td> | + | <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>P<i>luxR</i></td> | + | <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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<img class= "img-responsive" src="https://static.igem.org/mediawiki/2014/1/1f/Pai_blotsss.png" /> | <img class= "img-responsive" src="https://static.igem.org/mediawiki/2014/1/1f/Pai_blotsss.png" /> | ||
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| - | As shown in Fig 3A, GFP production is activated by PAI-1 following a 60 minute induction period. Thus the | + | 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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| - | <td>P<i>tet</i>-<i>lasR</i>- | + | <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>P<i>tet</i>-<i>lasR</i>- | + | <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>P<i>tet</i>-<i>lasR</i>- | + | <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>P<i>tet</i>-<i>lasR</i>- | + | <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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| - | <a href="pqs" class="btn btn-default toLesson">Previous Lesson: PQS</a> | + | <a href="pqs" class="btn btn-default toLesson">Previous Lesson: PQS Sensor</a> |
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| - | <a href="bdsf" class="btn btn-default toLesson">Next Lesson: BDSF</a> | + | <a href="bdsf" class="btn btn-default toLesson">Next Lesson: BDSF Sensor</a> |
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Latest revision as of 22:21, 17 October 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.
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
Welcome to Dundee's 2014 Wiki (Site Information)
Cystic Fibrosis is a genetic disease that results in the accumulation of thick mucus in the epithelial linings of the entire body, particularly the respiratory tract. Over the course of a patient’s life, the mucus-lined lung epithelium becomes repeatedly infected with pathogenic bacteria that stimulate an immune response; leading to inflammation, tissue damage, and ultimately, respiratory failure.
The microflora of the Cystic Fibrosis lung changes over time. In childhood, the major coloniser is Staphylococcus aureus, but as the patient matures other bacterial pathogens infect. The later-dominating pathogens, Pseudomonas aeruginosa and Burkholderia cepacia, are very difficult to eradicate and are associated with chronic decline in lung function. Burkholderia is so infectious that patients have to be isolated from one another, and can be denied lung transplants due to the persistence of this bacterium.
It is important, therefore, for medics to monitor and identify the levels of bacteria within the respiratory tract of a Cystic Fibrosis patient. Currently, patients must provide sputum samples and identification of bacteria takes between 72 hours and 2 weeks, by which point the bacteria can be in an antibiotic resistant state.
The Dundee 2014 iGEM project is focused on designing and testing a device that will rapidly and non-invasively identify the bacteria colonising a Cystic Fibrosis patient. Three biosensors will be developed that recognise external signal molecules produced by key bacteria, all of which are known to be in sputum samples of Cystic Fibrosis patients. A quinolone signal (PQS) is produced by Pseudomonas aeruginosa, a Diffusible Signal Factor (DSF) is produced by Stenotrophomonas maltophilia, an organism that is also associated with Cystic Fibrosis lung infection in adults, and BDSF is a related, but chemically distinct molecule that is produced by Burkholderia cepacia.
By engineering signal recognition and signal transduction proteins from these biological systems the Dundee iGEM team want to produce a portable electronic device that will identify infection outside of the clinic, so that patients do not have to travel great distances, and can be used to help health professionals make informed and rapid decisions on antibiotic treatments.
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