Part:BBa_K4194015
p(TetR)-RBS-EGFP-LVA-RBS-pleD-LVA-terminator-p(Bla)-RBS-(c-di-GMP riboswitch)-mRFP1-LVA-RBS-TetR-LVA
This is a gene oscillator. The feedback loop of this gene oscillator consists of c-di-GMP riboswitch and Tet gene expression regulation system. Promoter p(Bla) itself is a medium-strength constitutive promoter, but its downstream c-di-GMP riboswitch can successfully start the downstream gene expression only when the c-di-GMP concentration is higher than a certain value, because the c-di-GMP riboswitch can specifically sense the intra-cellular c-di-GMP concentration, and when the c-di-GMP concentration increases, it is turned on. The intracellular c-di-GMP concentration is regulated by two enzymes with opposite functions, namely bicyclic acid ester cyclase (DGC) which synthesizes c-di-GMP and phosphodiesterase (PDE) which degrades c-di-GMP. Therefore, we can change the intracellular c-di-GMP level by expressing the pleD gene of bicyclic acid ester cyclase (DGC). Ribose switch is a RNA sequence in the 5'-untranslated region (5'-UTR) of mRNA, which includes the aptamer domain (AD), a conserved sequence that can recognize and bind to ligands, and the expression platform (EP), which has variable structure and can regulate the downstream coding genes. The c-di-GMP riboswitch may form a terminator structure, which shuts down the transcription of downstream genes without c-di-GMP. However, in the presence of c-di-GMP, no terminator will be formed. The Tet gene expression regulation system is based on the Tet resistance operon of E.coli Tn10 transposon. Tetra has specific affinity with the DNA sequence in TetO. Tetra binds to two tetracycline manipulation sites (TetO) in P (Tetra) with high affinity, and inhibits the transcription of downstream genes, which can be released by tetracycline. Therefore, we can pause the whole periodic oscillation by adding a certain amount of tetracycline. Tet gene expression regulation system is commonly used, which has abundant characterization data and is reliable. In addition, the Tet system has the characteristics of low background and high induction multiple. The expression level of the target gene is low when it is not induced, but it is increased when it is induced, and the highest induction multiple can reach 10,000 times. The dynamic regulation range is relatively large, so it is suitable for participating in the feedback loop of gene oscillator. In addition, in order to ensure the strong oscillation of the gene oscillator, it is necessary to degrade the proteins involved in the feedback in time. At the same time, the stability of fluorescent protein is unfavorable to the characterization of gene oscillator. Therefore, ssrA-LVA tag is added at the end of GFP, PleD, RFP and tetR. SsrA tag is a 9~11-length amino acid sequence attached to the end of protein. Protein linked by SsrA tag can be degraded well by some combinations of protein (clpX, clpP and sspB). According to Andersen J B et al.'s research, the half-life of mature GFP(LVA) in vivo is about 40 minutes, while as a control, the colony of E.coli expressing GFP still keeps green fluorescence after several weeks.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1359
Illegal NotI site found at 1508
Illegal NotI site found at 1611
Illegal NotI site found at 2258
Illegal NotI site found at 3282 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1597
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1811
Illegal AgeI site found at 3164 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2585
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