Part:BBa_K4192132
Plac-RBS-yf1(N37C)-RBS-fixJ-PfixK2-RBS-mcherry
The gene yf1, fixJ and PfixK2 make up the system of blue light regulation. Under dark conditions, phosphate groups are transferred from Yf1 protein to FixJ protein, and the phosphorylated FixJ protein activates the PfixK2 promoter, which in turn activates downstream gene expression. Under light induction, the phosphorylation of Fixj protein was blocked and the expression of genes regulated by the PfixK2 promoter was suppressed.The mCherry protein emits red fluorescence to characterize the efficiency of control elements in regulating gene expression. We mutated position 37 N of YF1 to C to improve its response to blue light and better regulate gene expression.
Usage
Light control system can regulate gene expression by light intensity. MCherry has an excitation wavelength of 580nm and an emission wavelength of 610nm. Quantitative detection of mCherry can be achieved by detecting the optical signal at 610nm. This composition part examines the ability of the light control system containing the yf1 mutation to control gene expression and compares it with the normal light control system (BBa_ K4192130). The mutant yf1 (N37C) to expand the dynamic range of regulated gene expression under different signal intensities of the light control system, which would be more conducive to the abundant expression of downstream genes under dark conditions.
Biology
In the absence of light, FixL first undergoes autophosphorylation at histidine at position 29, and then transfers the phosphate moiety to its homologous, noncovalently bound response regulator FixJ. Phosphorylated FixJ binds to homologous promoters to activate transcription; In the presence of light, FixJ cannot be phosphorylated, no longer binds to the promoter, and transcriptional activity is downregulated.
Fig.1 the principle of light control system
The N37C mutation is located at a residue near the nonpolar dimethylbenzene portion of the flavin chromophore. The N37C mutation has a beneficial effect on regulation, that is, more target genes can be expressed under light conditions, and leakage expression can be minimized under light conditions. Specifically, the mutation of N37C in the LOV domain does not affect the way it responds to light and accelerates the dark recovery rate[1]. Compared with other mutations, which have unnecessary and adverse effects on the regulation of light and lead to the reduction of absolute light responsiveness, N37C mutation has little effect on absolute light responsiveness, that is, it does not affect the apparent quantum yield [2].
References
- Raffelberg, S., et al., The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein. bchm, 2013. 394(11): p. 1517-1528.
- Diensthuber, R.P., et al., Biophysical, Mutational, and Functional Investigation of the Chromophore-Binding Pocket of Light-Oxygen-Voltage Photoreceptors. ACS Synthetic Biology, 2014. 3(11): p. 811-819.
Sequence and Features
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Illegal AgeI site found at 1618 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1507
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