Team:Tokyo-Nokogen/Project/Light-receptor
From 2009.igem.org
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<h3><p style="margin-left:50px; margin-right:50px">[METHOD]<br> | <h3><p style="margin-left:50px; margin-right:50px">[METHOD]<br> | ||
(1) Design of the green light receptor | (1) Design of the green light receptor | ||
- | First, we identified the sequences of the red light responsive domain (cph1) and EnvZ domain in cph8 by BLAST search. We also searched the light responsive domains in CcaS (Fig. 2). The fusion protein of the light responsive domains of CcaS and EnvZ was designed by changing the histidine kinase domain of CcaS to EnvZ domain of cph8.<br> | + | First, we identified the sequences of the red light responsive domain (cph1) and EnvZ domain in cph8 by BLAST search. We also searched the light responsive domains in CcaS (Fig. 2). The fusion protein of the light responsive domains of CcaS and EnvZ was designed by changing the histidine kinase domain of CcaS to EnvZ domain of cph8.<h3><br> |
<p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/9/96/2-1.png"></p><br> | <p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/9/96/2-1.png"></p><br> | ||
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<h3><p style="margin-left:50px; margin-right:50px">(2) Construction of green light receptor (a new part) | <h3><p style="margin-left:50px; margin-right:50px">(2) Construction of green light receptor (a new part) | ||
We requested to synthesize two fragments of the photo-responsive domain of CcaS to Mr. Gene. The 1st fragment was designed containing an <I>Eco</I>RI and <I>Xba</I>I site on the N-terminal region and an <I>Nhe</I>I site at the C-terminus. The 2nd fragment was also designed containing <I>Eco</I>RI, <I>Xba</I>I and <I>Nhe</I>I sites on its N-terminal region and an <I>Nde</I>I site at its C-terminus. The cph8 gene, constructed by Jeff Tabor (UTAustin 2004), contained an <I>Nde</I>I site at the start of the EnvZ domain. | We requested to synthesize two fragments of the photo-responsive domain of CcaS to Mr. Gene. The 1st fragment was designed containing an <I>Eco</I>RI and <I>Xba</I>I site on the N-terminal region and an <I>Nhe</I>I site at the C-terminus. The 2nd fragment was also designed containing <I>Eco</I>RI, <I>Xba</I>I and <I>Nhe</I>I sites on its N-terminal region and an <I>Nde</I>I site at its C-terminus. The cph8 gene, constructed by Jeff Tabor (UTAustin 2004), contained an <I>Nde</I>I site at the start of the EnvZ domain. | ||
- | The 1st fragment was digested with <I>Eco</I>RI and <I>Nhe</I>I. The 2nd fragment was digested with <I>Nhe</I>I and <I>Nde</I>I, and the cph8 (BBa_I15010) was also digested with <I>Nde</I>I and <I>Pst</I>I. All these digested samples were ligated in a single tube with a BioBrick plasmid backbone digested with <I>Eco</I>RI and <I>Pst</I>I. (Fig. 3) | + | The 1st fragment was digested with <I>Eco</I>RI and <I>Nhe</I>I. The 2nd fragment was digested with <I>Nhe</I>I and <I>Nde</I>I, and the cph8 (BBa_I15010) was also digested with <I>Nde</I>I and <I>Pst</I>I. All these digested samples were ligated in a single tube with a BioBrick plasmid backbone digested with <I>Eco</I>RI and <I>Pst</I>I. (Fig. 3)<h3> |
<p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/4/44/3-1.png"></p><br> | <p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/4/44/3-1.png"></p><br> | ||
<h3><p style="margin-left:50px; margin-right:50px">(3) Construction of the green light activated actuator | <h3><p style="margin-left:50px; margin-right:50px">(3) Construction of the green light activated actuator | ||
- | We tried to construct green light activated actuator composed of phycocyanobilin synthesis genes and green light receptor (Fig. 4). Unfortunately, we did not succeed to construct the entire actuator part so we could not yet confirm that it works as expected. However we do expect it to function because the amino acid sequences of the light responsive domain of CcaS and previously designed cph8 are similar and the fusion protein was constructed by simply changing the kinase domain of CcaS to EnvZ. | + | We tried to construct green light activated actuator composed of phycocyanobilin synthesis genes and green light receptor (Fig. 4). Unfortunately, we did not succeed to construct the entire actuator part so we could not yet confirm that it works as expected. However we do expect it to function because the amino acid sequences of the light responsive domain of CcaS and previously designed cph8 are similar and the fusion protein was constructed by simply changing the kinase domain of CcaS to EnvZ.<h3> |
<p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/b/ba/4-1.png"></p> | <p style="margin-left:70px"><img src="https://static.igem.org/mediawiki/2009/b/ba/4-1.png"></p> | ||
<h3><p style="margin-left:50px; margin-right:50px">[Reference]<br> | <h3><p style="margin-left:50px; margin-right:50px">[Reference]<br> | ||
- | [1] Hirose et al. (2008) Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. <I>Proc Natl Acad Sci U S A</I>.105(28), 9528-33</p><br><br> | + | [1] Hirose et al. (2008) Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. <I>Proc Natl Acad Sci U S A</I>.105(28), 9528-33</p><h3><br><br> |
<img src="https://static.igem.org/mediawiki/2009/6/63/Red1.png" style="margin-left:45px"><div style="margin-left:45px; margin-right:15px"> | <img src="https://static.igem.org/mediawiki/2009/6/63/Red1.png" style="margin-left:45px"><div style="margin-left:45px; margin-right:15px"> | ||
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<h3><p style="margin-left:50px; margin-right:50px">[overview]<br> | <h3><p style="margin-left:50px; margin-right:50px">[overview]<br> | ||
In a previous iGEM competition, Levskyaya et al1 designed a bacterial system that is switched between different states by red light. In this system, phycocyanobilin biosynthetic genes (BBa_I15008 and BBa_I15009) for chromophore formation and the fusion of the red-light-responsive domain with the EnvZ histidine kinase domain (BBa_I15010), which phosphorylates endogenous OmpR as the second signal, have been used as red light sensing parts (Fig. 1). In order to regulate our ESCAPE system, we tried to construct a red-light receptor using the chimeric Cph1 light receptor /EnvZ protein (Cph8)1. | In a previous iGEM competition, Levskyaya et al1 designed a bacterial system that is switched between different states by red light. In this system, phycocyanobilin biosynthetic genes (BBa_I15008 and BBa_I15009) for chromophore formation and the fusion of the red-light-responsive domain with the EnvZ histidine kinase domain (BBa_I15010), which phosphorylates endogenous OmpR as the second signal, have been used as red light sensing parts (Fig. 1). In order to regulate our ESCAPE system, we tried to construct a red-light receptor using the chimeric Cph1 light receptor /EnvZ protein (Cph8)1. | ||
- | The part of the photoreceptor that responds to light, phycocyanobilin, is not naturally produced in <I>E. coli</I>. We therefore introduced two phycocyanobilin-biosynthesis genes, ho1 (BBa_I15008) and pcyA (BBa_I15009) from <I>Synechocystis</I> that convert heme into phycocyanobilin8 as described previously1.<br><br> | + | The part of the photoreceptor that responds to light, phycocyanobilin, is not naturally produced in <I>E. coli</I>. We therefore introduced two phycocyanobilin-biosynthesis genes, ho1 (BBa_I15008) and pcyA (BBa_I15009) from <I>Synechocystis</I> that convert heme into phycocyanobilin8 as described previously1.<h3><br><br> |
[Method]<br> | [Method]<br> | ||
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<h3><p style="margin-left:50px; margin-right:50px">[Results and discussion]<br> | <h3><p style="margin-left:50px; margin-right:50px">[Results and discussion]<br> | ||
- | We constructed and confirmed the sequences of the following three parts:<br> | + | We constructed and confirmed the sequences of the following three parts:<h3><br> |
<p style="margin-left:30px"><img src="https://static.igem.org/mediawiki/2009/3/31/16-1.png"></p> | <p style="margin-left:30px"><img src="https://static.igem.org/mediawiki/2009/3/31/16-1.png"></p> | ||
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<h3><p style="margin-left:50px; margin-right:50px">[Reference]<br> | <h3><p style="margin-left:50px; margin-right:50px">[Reference]<br> | ||
- | 1. Levskyaya et al, <I>Nature</I>, 438, 441-442 (2005), Engineering <I>Escherichia coli</I> to see light <br><br><br> | + | 1. Levskyaya et al, <I>Nature</I>, 438, 441-442 (2005), Engineering <I>Escherichia coli</I> to see light <h3><br><br><br> |
Revision as of 01:33, 22 October 2009