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 P_cblD-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 P_manA-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.

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