Project

  Introduction

Cell signaling and the quorum sensing is mediated by concentrations of the autoinducers PAI-1 and PAI-2, which is why their mere presence indicates existence of P. aeruginosa^[1]. The production of PAI-1 and PAI-2 are specific to only the las and the rhl systems respectively^[2]. Transcriptional activation of the target gene can be amplified up to 1000-fold due to the binding of the R-protein-autoinducer dimer complex and the induced promoter^[3]. Therefore, detecting the presence of P. aeruginosa can be reliant upon the detection of the autoinducers PAI-1 and PAI-2. Our detection system uses the specificity of the autoinducers as a definitive means of detecting P. aeruginosa. This approach focuses primarily on the robust detection of P. aeruginosa, without affecting the pathogen itself.

Figure 1: Activation of the induced promoters of P. aeruginosa Las and Rhl sequences by autoinducer/R-protein complexes (transcriptional regulation)

The detection system incorporates the use of the LasR and RhlR as the cognate R-proteins of the autoinducers PAI-1 and PAI-2. As strongly supported by experimental evidence, LasR/PAI-1 and RhlR and PAI-2 protein complexes act primarily as transcriptional factors^[4]. Therefore, our construct will be equipped with inducible promoters extracted from the genome of P. aeruginosa as indicated in Figure 2. These promoters are complementary to the dimer protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. The sole purpose of the inducible promoters is to facilitate the production of a reporting protein such as GFP and/or RFP as a means of P. aeruginosa detection.

Figure 2: Coupling the induced promoters of P. aeruginosa with reporting constructs (inserts 1 and 2)

In our construct, the lasR and rhlR gene will be paired with an upstream high efficiency constitutive promoter and ribosome binding site (RBS). Over time, constitutively encoded LasR and RhlR proteins will saturate the cell. Cell signaling autoinducers – PAI-1 and PAI-2 – will be the rate limiting reagents of the reporting genes’ biochemical induction process. The rate of binding of the autoinducers to their cognate R-proteins outpaces the rate of autoinducer degradation. Therefore upon the release of PAI-1 and PAI-2, our engineered E. coli will immediately commence the reporting process.