Difference between revisions of "Part:BBa K1981101"
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[[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 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.]]
 
[[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 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.]]
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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, amilCP (BBa_K592009).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.
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[[Image:AI-2 Response Device A|600px|thumb|center|'''Figure 1:''' Schematic overview of AI-2 Response Device. This device uses AI-2 inducible promoter lsr to induce GFP expression ]]
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===Characterization===
 
===Characterization===

Revision as of 00:10, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

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, amilCP (BBa_K592009).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.

File:AI-2 Response Device A
Figure 1: Schematic overview of AI-2 Response Device. This device uses AI-2 inducible promoter lsr to induce GFP expression


Characterization

AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.

Figure 2: Colony PCR Verification for AI-2 Response Device A.

AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.

Figure 1: GFP expression of AI-2 Response Device A when add exogenous AI-2.