Initial Planning and Cloning Strategy

Pseudomonas quinolone 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 virulence¹. Expression of these traits is mediated through the LysR-type transcriptional regulator, PqsR. Bioinformatic analysis of the PqsR sequence predicted that it is a soluble protein. Fractionation experiments of P. aeruginosa cells has shown that PqsR is primarily associated with the inner membrane². It is not clear however, whether this is through protein-lipid or protein-protein interactions with membrane lipids or with a yet unidentified integral inner membrane protein, respectively. In the presence of PQS, PqsR interacts with the promoter region of the pqsA operon, allowing transcription of the downstream genes³. We have engineered E. coli to express this signal transduction system for the detection of PQS (as shown in Fig 1), with a promoter-less mCherry coding gene fused to the pqsA promoter to give a fluorescent output.

Chromosomal DNA from Pseudomonas aeruginosa PA01 strain 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 a template for the amplification of the pqsA promoter gene.

The pqsA promoter region was cloned into the pSB1C3 plasmid (BioBrick BBa_K1315001), and was then subcloned into pBluescript. Promoterless mCherry was amplified (template used: BBa_K562012), and was cloned into pBluescript downstream of the pqsA promoter. The P_pqsA-mCherry construct was then subcloned into pUniprom. A schematic of our completed construct can be seen in Fig 2.

The pqsR gene (present in BBa_K1315001) 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 by commercially available antibodies. The HA-tag was fused to the C-terminus of the protein to allow visualisation by immunohistochemistry.

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 recombinantely expressed protein. 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 100% DMSO) at finals concentrations of 50 and 500 μM. A western blot with anti-mCherry antibodies was performed on the treated cells alongside a control (DMSO). Two further controls - MC1061 cells harbouring the empty pUniprom vector (mCherry negative control) and 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. 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.