Difference between revisions of "Part:BBa K1981101"
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| − | 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). | + | 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 <i>E.coli</i> MG1655, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). <i>plsr</i> is the promoter of the <i>lsr</i> operon. In our project, we use it to trigger the expression of a reporter gene，GFP. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | + | AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by <i>E.coli</i> MG1655, AI-2 response involves <i>lsr</i> gene clusters that encode <i>lsrACDB</i>, <i>lsrK</i>, <i>lsrFG</i>. <i>plsr</i> is the promoter of the <i>lsr</i> operon. | |
| − | AI-2 | + | |
| − | [[Image:NKU China lsr Promoter 3 hour curve.png| | + | [[Image:NKU China AI-2-luxS signal pathway in E-coli.png|600px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 response pathway in <i>E.coli</i> 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 <i>lsr</i> operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.]] |
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| + | <i>plsr</i> 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. | ||
| + | [[Image:AI-2 Response Device A.png|400px|thumb|center|'''Figure 2:''' Schematic overview of AI-2 Response Device A. This device uses AI-2 inducible promoter <i>plsr</i> to induce GFP expression. ]] | ||
| + | [[Image:AI-2 Response Device B.png|400px|thumb|center|'''Figure 3:''' Schematic overview of AI-2 Response Device B. This device uses AI-2 inducible promoter <i>plsr</i> 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. ]] | ||
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| + | ===Characterization=== | ||
| + | We isolated the promoter region of the <i>lsr</i> operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the <i>lsr</i> promoter, we transformed the plasmid pTrcHisB containing <i>plsr</i> with GFP gene at its downstream into <i>E. coli</i> 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. | ||
| + | [[Image:NKU China lsr Promoter 3 hour curve.png|500px|thumb|center|'''Figure 4:''' GFP expression after promoter <i>lsr</i> is induced by exogenous AI-2 .]] | ||
| − | AI-2 | + | Then we test whether promoter <i>lsr</i> 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 <i>lsr</i> can respond to different AI-2 concentration resulting in different GFP expression. |
| − | [[Image:NKU China exogenously Added AI-2 On DeviceA NKU China.png| | + | [[Image:NKU China exogenously Added AI-2 On DeviceA NKU China.png|500px|thumb|center|'''Figure 1:''' GFP expression when add exogenous AI-2.]] |
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
- 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.
