Difference between revisions of "Part:BBa K2012015"

Revision as of 11:12, 17 October 2016

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" )

00.1
Figure 1. Mechanism of CcaS-CcaR system.

CcaS is produced in a green-absorbing ground state, termed Pg. Absorption of green light flips CcaS to a kinase-active red-absorbing state (Pr) that phosphorylates the response regulator CcaR, which then binds to the G-box operator within cpcG2 promoter and activates transcription. Absorption of red light switches CcaS Pr back to Pg, which dephosphorylates P-CcaR, deactivating transcription.

Figure 3. 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.

More importantly, leaked expression in darkness significantly reduced after truncation of the constitutive promoter.

References:

1. 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. Proc Natl Acad Sci U S A 105: 9528-9533.
2. Tabor, J. J., Levskaya, A., and Voigt, C. A. (2011). Multichromatic control of gene''expression in Escherichia coli. J. Mol. Biol. 405, 2, 315–324.
3. Sebastian R. Schmidl, Ravi U. Sheth, Andrew Wu, and Jeffrey J. Tabor. Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synth. Biol. 2014, 3, 820−831

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]

@@ Line 25: / Line 25: @@
 <p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">&nbsp;</span></p>
-<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><strong>Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as </span><span style="font-family:Calibri">chromophore</span></strong><span style="font-family:Calibri">. </span><span style="font-family:Calibri"><span></span></span></p>
+<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><strong>Figure 3. Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as </span><span style="font-family:Calibri">chromophore</span></strong><span style="font-family:Calibri">. </span><span style="font-family:Calibri"><span></span></span></p>
 <p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">F</span><span style="font-family:Calibri">luorescence under green light is 1.81-folds of red light, proving that green light activates output expression, the device works well. </span><span style="font-family:Calibri">PcpcG2-172 </span><span style="font-family:Calibri">shows high efficiency.<span></span></span></p>
 <p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">More importantly, l</span><span style="font-family:Calibri">eaked expression in darkness significantly reduced after truncation of the constitutive promoter. </span><span style="font-family:Calibri;font-weight:bold"><span></span></span></p>