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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 | + | 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 E. coli chassis was responsible for activating GFP production, either via phosphorylation of BCAM0228 or by directly activating the cblD promoter, we made a new construct harbouring cblD-gfp and BCAM0228-HA, but lacking BCAM0227. 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 E. coli promoting the phosphorylated state of the BCAM0228 response regulator. | + | 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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| - | We are continuing to investigate this issue by screening E. coli mutants carrying deletions in genes coding for sensor kinases, taking advantage of the E. coli Keio mutant | + | 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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Revision as of 20:17, 4 October 2014
'Bold text'
Project
What we did
The Pseudomonas Quinolone Signal (PQS) sensing system: What it is and how it works
Initial planning and cloning strategy
Pseudomonasquinolone signal (2-heptyl-3-hydroxy-4-quinolone) is a quorum-sensing molecule produced by Pseudomonas aeruginosa, which regulates the expression of genes involved in biofilm development and virulence.1 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 P. aeruginosa has shown that the protein is primarily associated with the inner membrane. 2 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 pqsABCDE operon, allowing transcription of the downstream genes.3 We have engineered E. coli to express this signal transduction system for the detection of PQS, with a promoter-less mCherry fused to the pqsA promoter to give a fluorescent output.
Building the PQS sensor
Chromosomal DNA from Pseudomonas Aeruginosa 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 pqsA promoter. The pqsA 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 pqsABCDE promoter. The PpqsA-mCherry construct was then subcloned into pUniprom. 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 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.
The completed construct was transformed into E. coli strain MC1061 as a chassis for our biosensor.
Characterisation
Initially, western blots were undertaken to test for the production of PqsR-HA. We transformed our plasmid encoding PqsR-HA into E. coli 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.
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.
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 E. coli chassis. We therefore fractionated E. coli 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.
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.
The Burkholderia Diffusible signalling Factor (BDSF) sensing system: What it is and how it works
Initial planning and cloning strategy
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. Burkholderia Diffusible Signalling Factor (BDSF) is a Cis-2 dodecenoic acid that is produced exclusively by the pathogenic bacteria of the Burkholderia cepacia complex where it regulates expression of genes involved in virulence1,2. It is structurally similar to, but distinct from, DSF (cis-11-methyl-2-dodecenoic acid) produced by Stenotrophomonas maltophilia. In B. cenocepacia 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 cblD, a gene involved in Burkholderia virulence2. We engineered E. coli to express this signal transduction system for the detection of BDSF, with a promoter-less gfp gene downstream of the cblD promoter. With this, our sensor will detect any BDSF in its environment via the BCAM0227 receptor and activate GFP production.
Building the BDSF sensor
Chromosomal DNA from Burkholderia cenocepacia 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. The cblD promoter region was cloned into the pSB1C3 plasmid (to give Biobrick BBa_K1315008), and was then subcloned into pBluescript. Promoterless gfp was amplified using BBa_K562012 as a template, and was cloned into pBluescript downstream of the cblD promoter. The manA promoter-gfp construct was then subcloned into pUniprom. 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 PcblD-gfp. To adhere to the iGEM rules and regulations, it was necessary to remove an illegal EcoRI restriction site present in BCAM0228. The modified gene was cloned into the pSB1C3
plasmid (to give Biobrick BBa_K1315007). BCAM0228 was then subcloned into the pUniprom vector that already harboured PmanA-gfp and BCAM0227. 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. The plasmid was verified by sequencing. The completed construct was transformed into MC1061 E. coli as a chassis for our biosensor.
Characterisation
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.
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 Burkholderia3 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.
These results indicate that GFP production is activated regardless of the presence of BDSF. To test whether our E. coli chassis was responsible for activating GFP production, either via phosphorylation of BCAM0228 or by directly activating the cblD promoter, we made a new construct harbouring cblD-gfp and BCAM0228-HA, but lacking BCAM0227. 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 E. coli promoting the phosphorylated state of the BCAM0228 response regulator.
We are continuing to investigate this issue by screening E. coli mutants carrying deletions in genes coding for sensor kinases, taking advantage of the E. coli Keio mutant collection3. 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 pta-ackA double deletion mutant. Further work is required before the BDSF sensor is fully functional. The Following parts were deposited as Biobricks
| Part | Description | Registry |
|---|---|---|
| BCAM0228 | BDSF receptor/histidine kinase | BBa_K1315007 |
| CblD | BCAM0228 inducible promoter | BBa_K1315008 |
