Part:BBa_K1981101
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
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.
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.
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.
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