Difference between revisions of "Part:BBa K592004"

Latest revision as of 16:54, 9 October 2022

YF1 blue-light sensor protein

YF1 is a fusion protein of YtvA (B subtilis) and FixL (B japonicum). It contains the N-terminal region of YtvA (1-127) and C-terminal region of FixL (258-505), interconnected by a helical linker. This fusion protein is designed by Andreas Moglich and Keith Moffat. In their article (see references) they showed that illumination of the fusion kinase YF1 reduced net kinase activity by ∼1000-fold in vitro. They also showed that YF1 also controls gene expression in a light-dependent manner in vivo.

References

Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.

Contribution From CAU_China 2022

Group: CAU_China, 2022 https://2022.igem.org/Team:CAU_China

Author: Li Jiahui

Summary: Added information about how this part response to light control and improvement.

Biology

YF1 is a fusion protein coming from the heme-binding PAS sensor domain of FixL from Bradyrhizobium japonicum being replaced by light-oxygen-voltage (LOV) blue light sensor domain of Bacillus subtilis YtvA.

Fig.1 Schematic diagram of YF1 construction^[1] .

The fusion proteins receptor are regulated by light instead of by oxygen. Yf1 includes a light sensor subdomain and an effector subdomain. The light sensor binds a flavin chromophore. It absorbs light and influences effectors to be biologically active. 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. The YF1-containing light control system was introduced into the engineered bacteria to make them light-responsive and could be manipulated by light in a spatially and temporally accurate, reversible and non-harmful way^[1].

Fig.2 Regulatory mechanism of YF1 construction^[1].

Improvement

CAU_China 2022 used this fusion protein to regulate downstream oxalate secretion with light. In the process of searching the literature, we found that a point mutation that can improve the regulatory efficiency was found, and we tried to construct a test circuit to characterize the mutated YF1 as an improved part^[3]. Although did not get results we expected because of time limitation, but we think sharing this information may benefit future teams.

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^[2]. 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^[3].

Fig.3 Activity measurements of YF1 variants with the pDusk-DsRed reporter system. For wild-type YF1 blue light (white bars) induces a∼15-fold repression of DsRed reporter fluorescence compared to dark conditions (black bars)^[2].

In our experiments, ytvA, fixJ fixL, PfixK2 and mCherry parts were obtained by genome extraction and PCR amplification. These parts were assembled into RBS-yf1-RBS-fixJ-PfixK2-RBS-mCherry through multi-step overlap PCR (RBS was introduced through primers). The assembled parts were combined with linearized pUC18 vector by Gibson, and the sequences were verified by sequencing.

Fig.4 Genomic circuit of BBa_K4192130, which is same as BBa_K4192132.

We designed primers with mutations for PCR amplification of pUC18 plasmid with RBS-yf1-RBS-fixJ-PfixK2-RBS- mcherry and selected mutants using Dpn I, an enzyme that can digest methylated and hemimethylated template DNA. Sequencing results showed that the N37C mutation was successfully introduced. However, we have been working on the measurement of reporter genes until Wiki Freeze and we assume the reason of our failure may because the _mCherry_ cannot express as we expected.

References of CAU_China

[1] Möglich, A., R.A. Ayers and K. Moffat, Design and Signaling Mechanism of Light-Regulated Histidine Kinases. Journal of Molecular Biology, 2009. 385(5): p. 1433-1444.

[2] 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.

[3] 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