Difference between revisions of "Part:BBa K2244009:Design"
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LEV1 is a light repressor with an E. coli lexA DNA-binding domain, which binds to its cognate operator sequence on promoter when dimerized upon light irradiation. LEV1 is a fusion protein of VVD and LexA: VVD contains a light-oxygen-voltage (LOV) domain & N cap was added to the N-terminal of DNA-binding domain of LexA. The C-terminal domain of lexA repressor was removed. The expression of Lev1 is under a constitutive promoter so that it is constantly expressed. | LEV1 is a light repressor with an E. coli lexA DNA-binding domain, which binds to its cognate operator sequence on promoter when dimerized upon light irradiation. LEV1 is a fusion protein of VVD and LexA: VVD contains a light-oxygen-voltage (LOV) domain & N cap was added to the N-terminal of DNA-binding domain of LexA. The C-terminal domain of lexA repressor was removed. The expression of Lev1 is under a constitutive promoter so that it is constantly expressed. | ||
| − | ColE promoter, with an intrinsic RBS, is obtained from E. coli | + | ColE promoter, with an intrinsic RBS, is obtained from E. coli ColE1 plasmid and contains a cognate operator sequence for LexA. Expression of target gene (in this case, mCherry) is under the control of ColE promoter, thus is also regulated by LEV1 binding and light irradiation. mCherry sequence is codon optimized for E. coli expression. T1 terminator is the most commonly used terminator in E. coli. |
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Latest revision as of 03:53, 2 November 2017
ColE promoter+mCherry gene+T1 terminator+Constitutive promoter+Lev1 gene+T1terminator
- 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]
Source
LEV1 is a light repressor with an E. coli lexA DNA-binding domain, which binds to its cognate operator sequence on promoter when dimerized upon light irradiation. LEV1 is a fusion protein of VVD and LexA: VVD contains a light-oxygen-voltage (LOV) domain & N cap was added to the N-terminal of DNA-binding domain of LexA. The C-terminal domain of lexA repressor was removed. The expression of Lev1 is under a constitutive promoter so that it is constantly expressed.
ColE promoter, with an intrinsic RBS, is obtained from E. coli ColE1 plasmid and contains a cognate operator sequence for LexA. Expression of target gene (in this case, mCherry) is under the control of ColE promoter, thus is also regulated by LEV1 binding and light irradiation. mCherry sequence is codon optimized for E. coli expression. T1 terminator is the most commonly used terminator in E. coli.