Difference between revisions of "Part:BBa K2012015"
 
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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" )
 
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" )
 
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<html><img alt="00.1" src="https://static.igem.org/mediawiki/parts/7/71/T--HZAU--China--CcaS-CcaR.jpg" style="float:none;width:400px;height:240px"></html>
 
 
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</span><strong>Figure 1. Mechanism of CcaS-CcaR system<span>.</span></strong><span><span>&nbsp;<br/>
 
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<span>&nbsp;</span></span></span></span><span>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.</span>
 
  
 
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<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">&nbsp;</span></p>
  
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<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>
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><strong>Figure 1.</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><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">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>
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<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>
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<span><h2>References:</h2></span><br/>
 
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<span>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.<br/>
 
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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.<br/>
 
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3. Sebastian&nbsp;R.&nbsp;Schmidl,&nbsp;Ravi&nbsp;U.&nbsp;Sheth,&nbsp;Andrew&nbsp;Wu,&nbsp;and&nbsp;Jeffrey&nbsp;J.&nbsp;Tabor.&nbsp;Refactoring&nbsp;and&nbsp;Optimization&nbsp;of&nbsp;Light-Switchable&nbsp;Escherichia&nbsp;coli&nbsp;Two-Component&nbsp;Systems.&nbsp;ACS&nbsp;Synth.&nbsp;Biol.&nbsp;2014,&nbsp;3,&nbsp;820−831</span></p>
 
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===Usage and Biology===
 
  
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<b>Improvement</b><br>A truncated promoter PcpcG2-68 has the similar pattern under the control of green/red light as PcpcG2-172, but is shorter and more convenient to construct. For example, when constructing an array of sgRNAs, PcpcG2-68 has a big advantage over PcpcG2-172: reducing the volume of the circuit and cheaper to synthesis.<br> [[File:T--XHD-Wuhan-B-China--111(1).png|900px|thumb|left|]]<br>Please view https://parts.igem.org/Part:BBa_K3921000 for more details.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2012015 parameters</partinfo>
 
<partinfo>BBa_K2012015 parameters</partinfo>
 
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<span id="IGEM 2021 XHD- Wuhan-B-China"><br><br><br><br><br></span>

Latest revision as of 06:41, 21 October 2021


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.




Improvement
A truncated promoter PcpcG2-68 has the similar pattern under the control of green/red light as PcpcG2-172, but is shorter and more convenient to construct. For example, when constructing an array of sgRNAs, PcpcG2-68 has a big advantage over PcpcG2-172: reducing the volume of the circuit and cheaper to synthesis.
T--XHD-Wuhan-B-China--111(1).png

Please view https://parts.igem.org/Part:BBa_K3921000 for more details.

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]