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 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.

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_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 with commercial antibodies. This tag was added to the C-terminus of the protein to facilitate 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 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.