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Quorum sensing was first found in Vibrio fischeri in the ocean. Microorganisms achieve information communication by releasing a signaling molecules referred as auto inducer(AI). AI will promotes related gene expression in bacteria, meanwhile regulate microbes’ biological behaviors. Bacteria use quorum sensing to coordinate certain behaviors such as biofilm formation, virulence, bioluminescence and antibiotic resistance, based on the local density of bacteria. Thus microbes can collaborate to achieve cell populations’s engineering.

In Vibrio fischeri , plux promoter regulates the expression of quorum sensing system while plux promoter is automatically regulated by AI. PluxL and pluxR respectively promote transcription to the left and right. PluxL regulates transcription of LuxR gene ;in the meantime, pluxR can also start expression of luxI and fluorescence related genes. LuxI, is responsible for production of N-acyl-homoserine-lactone (AHL) autoinducer while luxR,is activated by this autoinducer to increase transcription of the luciferase operon.

The enhanced E.coli quorum sensing system we reconstructed is greatly dependent to plac promoter. Plac will serve as pluxL in this system to promote transcription. Once the transcription has been started by plac, it will extend to series genes such as luxR、pluxR and luxI downstream. Noticeably, the LuxI/LuxR genes form a functional pair, considering LuxI as the auto-inducer synthase and LuxR as the receptor. We can obtain translation products luxI to catalyze the formation of AHL.

The yield of luxI protein is very low with a tenuous concentration of cells, likewise result in the subtle concentration of AHL in periplasmic. As the amount of cells increases, the density of AHL will reach a threshold value, this can strongly accelerate the formation of one kind of complex which is composed of AHL and luxR , also the complex will firmly promotes transcription of pluxR ,hence we acquire desired expression of bioluminescence related genes as well as increasing amount of luxI、luxR、AHL, which in turn form a positive regulation. In addition, plac is a fully functional part which act as a substitution to pluxL, preventing pluxL from inhibition of the AHL-LuxR complex, what’s more, releasing the negative influence on the expression of LuxR.

Based on this enhanced E.coli quorum sensing system, we got a innovative method for specific metal ions detection. The highlight is that we combine MerR and relevant promoter sequence pMerT to the downstream of luxI. The MerR family of metal-binding, metal-responsive proteins is unique in that they activate transcription from unusual promoters and coordinate metals through cysteine residues. They have conserved primary structures yet can effectively discriminate metals in vivo. Expression of MerR (in the absence of MerR and Hg (II) in the cell) proceeds from the PmerR promoter. In the absence of Hg(II), the MerR homodimer binds to the operator region within the divergent promoter with binding centered on the dyad symmetrical DNA sequence between the -35 and -10 sequences of PmerT, slightly repressing transcription of the structural gene promoter, and repressing transcription of merR from the PmerR promoter. Once the MerR homodimer has bound to merOP, recruitment of RNAP to the mer promoter occurs , and MerR has been shown to cross-link to several subunits of RNAP .

In the presence of mercuric ions, one Hg(II) per MerR homodimer coordinates in a trigonal manner to three essential cysteine residues of the MerR homodimer, two cysteines from one monomer, and one from the other. Hg(II) binding to the MerR homodimer results in both a relaxation of the DNA bends induced by apo-MerR, and both DNA distortion and an allosteric under winding of the promoter sequence by approximately 33. The under winding of the promoter DNA aligns the -10 and -35 sequences, such that RNA polymerase can recognize and bind to these sites, initiating transcription from PmerT .

The reporter gene we apply is gfp ,which also depends on the transcription of the same promoter pMerT.

According to the design, we intended to make best use of the recombinant E.coli quorum sensing system to increase the bacteria density to a threshold value and produce a sufficient amount of AHL and LuxR protein, forming a continuous positive feedback. Meanwhile, there supposed to be a “switch” referred as MerR and pMerT. Only the presence of Hg(II) do drive the expression of structural genes and fluorescence related gene. Moreover, the fluorescence intensity should theoretically link a certain correlation to the concentration of Hg(II) in internal condition. For the purpose of continuous detection, what should not be ignored is that we desire enough MerR homodimer to bind to pMerT then interdict the transcription pathway. This clarifies the second aspect of recombinant E.coli quorum sensing system. Concluded, we designed this innovative system intending to get a fasten detection method towards specific metal ions in vivo.