Difference between revisions of "Part:BBa K2244005"
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===short description===
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LEV1 is a coding gene encodes fusion protein LexA-VVD. VVD is a blue light sensor, a small light-oxygen-voltage (LOV) domain-containing protein from Neurospora crassa, and it is fused to the C-terminus of DNA-binding domain of LexA repressor of the Escherichia coli SOS regulon to form LEV1 fusion protein.
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LEV1 is a coding gene encodes fusion protein LexA-VVD  
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===long description===
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===Usage and Biology===
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This part is a light-switchable repressor component functions in E. coli cells. A blue light sensor VIVID (VVD), which is a small light-oxygen-voltage (LOV) domain-containing protein from Neurospora crassa, is fused to the C-terminus of DNA-binding domain of LexA repressor of the Escherichia coli SOS regulon. It’s a common feature for several blue light photoreceptors to contain LOV domains, which are able to bind a molecule of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as chromophore.
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Upon blue light irradiation, a cysteine flavin adduct is formed in the VVD domain, causing conformational changes of the domain and the subsequent dimerization of the fusion protein. The activated protein dimer would then bind its cognate operator sequence and repress the promoter activity. When in dark, the absence of dimer allows the proceeding of target gene expression. The LightOff system has high induction efficiency similar to T7-driven pET expression system, yet much lower leakage. This part works under E. coli SOS promoter and operator sequence.
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This fusion protein is a light repressor used in a single component light-repressed expression system. Upon blue light irradiation, a cysteine flavin adduct is formed in the VVD domain, causing conformational changes of the domain and the subsequent dimerization of the LEV1. The dimer would then bind its cognate operator sequence and repress the promoter activity. When in dark, the absence of dimer allows the proceeding of target gene expression. This fusion protein works with E. coli SOS promoter and associated operator sequence.
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<center><img src="https://static.igem.org/mediawiki/parts/b/b7/Opda.png" style=" width:80%" /> </center>;
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<center><b>Figure 1.</b> diagram of LEV1 fusion protein formation and dimerization </center>;
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Lev1 is 714bp. Figure 2 shows a colony PCR amplifying DNA section harboring ColE promoter and LEV1 gene in pLEV1(408) vector.
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Figure 2: The agarose gel electrophoresis of LEV1 colony PCR product
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===Reference===
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Chen, X., Liu, R., Ma, Z., Xu, X, Zhang, H., Xu, J. & Yang, 2016. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Research, 26 (7): 854-7.
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Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, et al. (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442.
  
 
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Revision as of 06:58, 27 October 2017

LEV1 repressor


LEV1 is a coding gene encodes fusion protein LexA-VVD. VVD is a blue light sensor, a small light-oxygen-voltage (LOV) domain-containing protein from Neurospora crassa, and it is fused to the C-terminus of DNA-binding domain of LexA repressor of the Escherichia coli SOS regulon to form LEV1 fusion protein.


Usage and Biology

This fusion protein is a light repressor used in a single component light-repressed expression system. Upon blue light irradiation, a cysteine flavin adduct is formed in the VVD domain, causing conformational changes of the domain and the subsequent dimerization of the LEV1. The dimer would then bind its cognate operator sequence and repress the promoter activity. When in dark, the absence of dimer allows the proceeding of target gene expression. This fusion protein works with E. coli SOS promoter and associated operator sequence.

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Figure 1. diagram of LEV1 fusion protein formation and dimerization
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Lev1 is 714bp. Figure 2 shows a colony PCR amplifying DNA section harboring ColE promoter and LEV1 gene in pLEV1(408) vector.







Figure 2: The agarose gel electrophoresis of LEV1 colony PCR product


Reference

Chen, X., Liu, R., Ma, Z., Xu, X, Zhang, H., Xu, J. & Yang, 2016. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Research, 26 (7): 854-7.

Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, et al. (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 677
  • 1000
    COMPATIBLE WITH RFC[1000]