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Team:Dundee/Project/PAI-1
From 2014.igem.org
Pseudomonas auto inducer-1 sensing system
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.
Building the PAI-1 sensor
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 |
Characterisation
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the construct were cultured in LB medium, cultures were spiked with 500μM synthetic PAI-1 in DMSO and samples were withdrawn at time periods of up to one hour following PAI-1 addition.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 |
