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 auto-inducer 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 promoter. Both2,3 PlasB and PluxR were adopted as LasB-PAI-1 inducible promoters in our device.2 Using pre-existing Biobricks we have designed new circuits to engineered E. coli to express the LasR transduction system for the detection of PAI-1, along with promoter-less gfp fused to either the lasB or luxR promoter.

All the parts we used to construct our PAI-1 sensor were obtained from the iGEM parts registry and sequentially cloned into pSB1C3 plasmid. The gene encoding the green fluorescent protein (GFP) was fused to our sensing device. Part Description Registry pTet TetR repressible promoter BBa_R0040 LasR CDS LasR activator from P. aeruginosa PAO1 BBa_C0179 LasR and PAI regulated promoter Binding region for LasR protein BBa_R0079 pluxR Promoter BBa_R0062 GFP Green fluorescent protein BBa_E0040 The plasmids were verified by sequencing. The completed construct was transformed into E. coli strain MC1061 as a chassis for our two PAI biosensors.

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the construct were cultured in LB medium, cultures were spiked with 500μM synthetic PAI-1 in DMSO and samples were withdrawn at time periods of up to one hour following PAI-1 addition.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 LasR/plasB 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 auto-inducer 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 LasB. 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