Part:BBa_K2012015

PcpcG2-172, a modified PcpcG2 promoter

PcpcG2 promoter is a 238bp green-light activated promoter in Synechocystis PCC 6803(Part:BBa_K592003). The full length promoter is comprised of a G-box region, a CcaR-P activated promoter, and a constitutive promoter, which contributes to the leakiness under red light and low dynamic range. Therefore, we constructed PcpcG2-172, a 172bp truncated cpcG2 promoter deleted for the constitutive promoter.(For more detail, please view our wiki: "http://2016.igem.org/Team:HZAU-China/Experiments" )

Figure 1. Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as chromophore.

Fluorescence under green light is 1.81-folds of red light, proving that green light activates output expression, the device works well. PcpcG2-172 shows high efficiency.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

iGEM 2021 XHD-Wuhan-B-China, new documentation (For Bronze)

Group: XHD-Wuhan-B-China

Author: Kening Chen

Summary: Measurement of PcpcG2-172 under new circumstances

PcpcG2-172 is a 172bp green-light induced promoter constructed by Sebastian R. Schmidl group (1).CcaS phosphorylates CcaR under green light and induces expression of cpcG2 (2).A study of Mitsuharu Nakajima group reveals that CcaS#10 works well when two PAS domains were deleted from the original sequence (3).Therefore, we tested PcpcG2-172 in a modified two-component system composed of CcaS#10 and a series of CcaRs with different transcription intensities.

Figure1. Light inducible CcaS-CcaR two component system. (A) Structure of the CcaS-CcaR circuit. (B) Measurement of PcpcG2-172 under red light (660nm) or green light (520nm). The experiment was carried out in MG1655-△EnvZ strain, using a 24-wall plate adapted LED device. Graphic shows that among four tested promoters, J23117-CcaR gives PcpcG2-172 best dynamic range of 8.3-fold change. When using stronger promoters J23114 and J23115, this dynamic range narrows as leakage under red light getting larger.

Figure2. Light-control devices used in this experiment.

Reference

[1] Schmidl SR, Sheth RU, Wu A, Tabor JJ. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol. 2014 Nov 21;3(11):820-31.
[2] Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A. 2008 Jul 15;105(28):9528-33.
[3] Nakajima M, Ferri S, Rögner M, Sode K. Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Sci Rep. 2016 Nov 24;6:37595.

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