Part:BBa_K2244009
ColE promoter+mCherry gene+T1 terminator+Constitutive promoter+Lev1 gene+T1terminator
The device is a functional plasmid containing a light repressor LEV1 (BBa_K2244005), which undergoes conformational change upon light irradiation and dimerized to bind regulatory sequence on ColE promoter (BBa_K2244006), thus inhibits target gene expression. While in darkness, dimerization does not occur thus gene expression proceeds. This device is a light repressing system with high induction efficiency and low leakage. mCherry ((BBa_K2244008)) is reporter gene used in this system to prove its function as well as help to characterize it. mCherry gene can be replaced by any gene sequence in light-regulated studies
Biology
ColE promoter(BBa_K2244006) sequence is derived from the promoter region of colicin E gene located in the ColE1 plasmid of E.coli. ColE promoter contains a ‘SOS’ operator region that allows the binding of LexA protein to repress transcription. mCherry is a red fluorescent protein used as a reporter. It is based on a fluorescent protein that was originally isolated from Discosoma sp.mCherry sequence is codon optimized for E. coli expressionm and can be replaced by any gene sequence in light-regulated studies.
LEV1 repressor(BBa_K2244005) is a fusion protein of VVD and LexA, LexA repressor is a transcriptional repressor of SOS regulon in E.coli. It’s form chromosome of Escherichia coli str. K-12 substr. MG1655 (strain: K-12, substrain: MG1655) LexA autocleavage, stimulated by RecA, of the first 84 aa of LexA removes the DNA binding region and is required to activate the SOS response. LexA is a protein that belongs to the LexA family .
Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation, Based on this property, the VVD LOV domain was fused with a smaller version of the Gal4 DNA binding domain and the p65 transactivation domain. A common feature of several blue light photoreceptors is the presence of LOV domains, which are able to bind a molecule of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as chromophore, forming upon light stimulation a cysteinyl flavin C4a adduct.
T1 terminator(BBa_B0010) is the most commonly used terminator in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1015
Illegal NheI site found at 1038 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 845
Illegal AgeI site found at 1753 - 1000COMPATIBLE WITH RFC[1000]
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