Team:NYMU-Taipei/ymis3.html

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            <h2 class="drawer-handle open">Sulfur Oxide Terminator</h2>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymis1.html">Overview</a></li>
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                <li><a title="Paper-Based Research" href="http://2012.igem.org/Team:NYMU-Taipei/ymis2.html">Paper-Based Research</a></li>
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                <li><a title="Experiment Design" href="http://2012.igem.org/Team:NYMU-Taipei/ymis3.html">Experiment Design</a></li>               
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                <li><a title="Practical Application in Industrial Waste Detection" href="http://2012.igem.org/Team:NYMU-Taipei/ymis5.html">Practical Applicatioin in <br />
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                <li><a title="Discussion" href="http://2012.igem.org/Team:NYMU-Taipei/ymis6.html">Discussion</a></li>
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                <li><a title="Conclusion & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymis7.html">Conclusion &amp; References</a></li>
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            <h2 class="drawer-handle">Sulfide as Energy Generator</h2>
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                <li><a title="Abstract" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq1.html">Abstract</a></li>
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                <li><a title="Methods" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
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                <li><a title="Measurements" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq3.html">Measurements</a></li>
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                <li><a title="Results & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Result</a>s &amp; References</li>
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            <h2 class="drawer-handle">Denitrifying Machine</h2>
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                <li><a title="Background" href="http://2012.igem.org/Team:NYMU-Taipei/ymin1.html">Background</a></li>
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                <li><a title="Methods" href="http://2012.igem.org/Team:NYMU-Taipei/ymin2.html">Methods</a></li>
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                <li><a title="Results" href="http://2012.igem.org/Team:NYMU-Taipei/ymin3.html">Results</a></li>
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                <li><a title="Practical Application & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymin4.html">Practical Application</a> &amp;<br />
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References</li>
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            <h2 class="drawer-handle">Cd+2 Collector</h2>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymic1.html">Overview</a></li>
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                <li><a title="Experiment Design" href="http://2012.igem.org/Team:NYMU-Taipei/ymic2.html">Experiment Design</a></li>
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                <li><a title="Methods & Materials" href="http://2012.igem.org/Team:NYMU-Taipei/ymic3.html">Methods & Materials</a></li>
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                <li><a title="Results & Discussion" href="http://2012.igem.org/Team:NYMU-Taipei/ymic4.html">Results & Discussion</a></li>
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                <li><a title="Conclusioin & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymic5.html">Conclusioin & References</a></li>
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            <h2 class="drawer-handle">Becoming Venusian</h2>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusian.html">Overview</a></li>
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                <li><a title="Introduction" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusianintro.html">Introduction</a></li>
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Revision as of 03:39, 27 September 2012

NYMU iGEM

Experiment Design

Gene Cloning of Cys I Sulfite Reductase

Firstly, we get the whole gene sequence of Cys I from NCBI web (www.ncbi.nlm.nih.gov/gene/878581). Cys I contain endogenous EcoRI and PstI site, so we can’t clone into PSB1C3 directly.As a result, We create a noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I) for easily cloning. Taking advantage of MfeI and EcoRI are compatible, also SbfI and XbaI, we can easily clone Cys I gene into pSB1C3 standard biobrick. Enzyme check by XbaI and SbfI, we can get ~1700bp band, which means our gene constraction is correct!!



In order to establish stable expression cyanobacteria system, we need to subclone our Cys I into stable expressing plasmid, pTrc-kan plasmid (constructed by Prof. Chang). pSB1C3-Cys I cut with MfeI and SPeI. pTrc-kan cut with EcoRI and XbaI. Finally we can get ~7100bp plasmid, pTrc-kan-Cys I gene. Enzyme check by SpeI and get ~2100bp and ~3000bp bands.


pTrc-kan-Cys I plasmid



Biobrick of Cys I sulfite reductase

Gene Cloning of Dsr Sulfite Reductase

As same as Cys I gene, we get the whole gene sequence of Dsr I and Dsr II gene
from NCBI web (http://www.ncbi.nlm.nih.gov/protein/CAC09931.1). DsrI and DsrII also contain endogenous EcoRI and PstI site, so we clone them into noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I). We create a BamH I site inside of DsrI and DsrII, in order to combine them into a whole part, “Dsr”. Enzyme check by XbaI and Spe I , we can get ~5800 bp, which means our gene constraction is correct!! Subclone our Dsr into stable expressing plasmid, pTrc-kan plasmid (constructed by Prof. Chang). pSB1C3-Dsr cut with MfeI and SPeI, pTrc-kan cut with EcoRI and XbaI. Finally we can get ~9800bp plasmid, pTrc-kan-Dsr gene. Enzyme check by BamHI, we can ~1200bp.




Biobrick of Dsr Sulfite Reductase