Green Light Sensing System

Previous studies have shown the reversible photoconversion between a green-absorbing form (Pg, λmax = 535 nm) and a red-absorbing form (Pr, λmax = 672 nm) of CcaS[1]. Autophosphorylation activity of the histidine kinase domain in the C-terminal region of CcaS is up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, is higher in Pr than in Pg. In cyanobacteria, CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. The chromophore needed in green light sensing system is phycocyanobilin(PCB),same with red sensing system, which is definitely good news.

Fg1. Two-color optical control of gene expression in E. coli. PMCID: PMC3053042

Downstream Regulation

Certain sgRNA is under control of the green light sensing system. sgRNA targeting RFP is cloned and the base pairing region was easily replaced by reverse PCR to target luciferase and FebI.

But there is a problem we cannot ignore, sgRNA transcripts must be strictly precise to function normally. Redundant deoxyribonucleoside in 5’-terminal of sgRNA may cause inactivation. Considering that we do not know transcription initiation site of CpcG2 and, more importantly,in order to be consistent with other light sensing systems, we did some modification to CcaR-responding promoter.

Previous studies have shown phosphorylated CcaR recognizes and binds to the G-box of CpcG2.[2]Six base pairs in the G-box are identical to -35 site of J23107(A constitutive promoter). So we overlap G-box to BBa_J23107. CcaR will bind to G-box, thus prevents RNA polymerase from binding to the promoter. This modification(BBa_K1023009) allows down-regulation effect under green light.

Fig.2 BBa_K1023009 design

Sensor Design

CcaR and CcaS was cloned from cyanobacteria and inserted into constitutive operon we have constructed respectively. Then, the integral of operon, CcaR and CcaS was cloned and inserted in pCDFDuet-1. sgRNA was inserted in opposite direction to avoid leaking expression from upstream terminator.

Fig.3 Final version of Green sensing system

Reference

HIROSE, Y., NARIKAWA, R., KATAYAMA, M. & IKEUCHI, M. 2010. Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter. Proceedings of the National Academy of Sciences, 107, 8854-8859.


HIROSE, Y., SHIMADA, T., NARIKAWA, R., KATAYAMA, M. & IKEUCHI, M. 2008. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences, 105, 9528-9533.


TABOR, J. J., LEVSKAYA, A. & VOIGT, C. A. 2011. Multichromatic control of gene expression in escherichia coli. Journal of Molecular Biology, 405, 315-324.