Part:BBa_K4194018
p(Bla)-Lac operator-RBS-CI-LVA-RBS-EGFP-LVA-terminator-pRM'-RBS-LacI-LVA-RBS-mRFP1-LVA-terminator
This is a gene oscillator.
The feedback loop of this gene oscillator consists of pRM' promoter (activated by CI protein) and Lac gene expression regulation system. Promoter p(Bla) was originally a medium-strength constitutive promoter, but the downstream lac operator could bind to LacI protein to repress promoter p(Bla). Promoter pRM' is an improved pRM promoter from λ phage (OR-3 is eliminated).
Phage λ is a virus that infects E.coli. After infecting the host E.coli cells, it enters two different life cycles: lytic cycle or lysogenic cycle. In λ phage, the dynamic balance of the separation or combination of CI protein and early transcription promoter pR controls the lysogenic or lytic pathway of phage. PRM is the promoter of CI protein, which maintains inhibitor, and only transcribes CI gene. The wild pRM promoter contains OR1, OR2 and OR3 sites, so it can be activated and inhibited by CI protein from the same phage. CI activates the transcription of this promoter at low concentration (binding to OR1 and OR2), but inhibits the transcription at high concentration (binding to OR3).
Under the condition of nutrient deficiency, cells accelerate the degradation of protein, so as to provide important nutrients for essential metabolic processes. The concentration of CI protein decreases, the pR promoter is released from inhibition, while pRM is in a closed state, and bacteria are in a cracking growth cycle. When pRM is in an open state and pR is in a closed state, bacteria are in a lysogenic growth cycle.
The improved promoter pRM' can be activated by CI protein, but not inhibited by CI protein.
In addition, in order to allow the protein to be degraded in time and make the feedback more sensitive, ssrA-LVA tag is added at the end of EGFP, CI, mRFP1 and LacI.
At the initial state of the system, there is no LacI protein and CI protein in the cell, and the P (Bla) promoter will open the expression of CI gene and EGFP gene, while pRM 'is not activated, so the downstream LacI gene and RFP gene are not expressed. With the expression of CI, the promoter pRM 'was activated, and the LacI gene and RFP gene were expressed. The presence of LacI inhibited promoter P (Bla), stopped the expression of CI and EGFP genes, and reduced the concentration of CI made the promoter pRM 'return to the low activity state. Promoter pRM 'was in the inactive state, RFP gene and LacI gene stopped transcription, and the system returned to the initial state. Thus realize independent dynamic regulation and control.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 2942
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 2942
Illegal NotI site found at 765
Illegal NotI site found at 3841 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1617
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2942
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2942
Illegal NgoMIV site found at 1649
Illegal NgoMIV site found at 2458
Illegal AgeI site found at 647
Illegal AgeI site found at 2035
Illegal AgeI site found at 3723 - 1000COMPATIBLE WITH RFC[1000]
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