Difference between revisions of "Part:BBa K1981101"

Latest revision as of 03:01, 15 October 2016

lsr promoter of LuxS/AI-2 signaling pathway in E.coli

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of E.coli MG1655, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). plsr is the promoter of the lsr operon. In our project, we use it to trigger the expression of a reporter gene，GFP.

Sequence and Features

Usage and Biology

AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by E.coli MG1655, AI-2 response involves lsr gene clusters that encode lsrACDB, lsrK, lsrFG. plsr is the promoter of the lsr operon.

Figure 1: Schematic overview of the AI-2 response pathway in E.coli MG1655. The precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione (DPD), is a byproduct generated when LuxS converts S-Ribosylhomocysteine (SRH) to Homocysteine (HCY). DPD then undergoes spontaneously cyclization, forming AI-2, and exports to the culture supernatant. After that, extracellular AI-2 bounds to LsrB, following by passing the membrane channel and importing the cytoplasm. LsrK phosphorylates AI-2 afterwards. The lsr operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.

plsr is an inducible promoter under the repressive regulation of LsrR. In our project, we use it to regulate the expression of the reporter gene, GFP.When pathogenic bacteria express AI-2 molecules and secrete them extracellularly, these AI-2 molecules can be transported into our engineered bacteria and trigger the expression of the report gene to produce the blue pigment.

Figure 2: Schematic overview of AI-2 Response Device A. This device uses AI-2 inducible promoter plsr to induce GFP expression.

Figure 3: Schematic overview of AI-2 Response Device B. This device uses AI-2 inducible promoter plsr to induce GFP expression. In AI-2 Response Device B, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response.

Characterization

We isolated the promoter region of the lsr operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the lsr promoter, we transformed the plasmid pTrcHisB containing plsr with GFP gene at its downstream into E. coli MG1655 delta luxS. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. As you can see, after induced by AI-2, the GFP expression is increased compared to control group.

Figure 4: GFP expression after promoter lsr is induced by exogenous AI-2 .

Then we test whether promoter lsr can respond to different AI-2 concentration. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that promoter lsr can respond to different AI-2 concentration resulting in different GFP expression.

Figure 1: GFP expression when add exogenous AI-2.