Team:NYMU-Taipei/ymic3.html

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       <span class="subtitle">Kill-switch of amoeba</span></div>
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     <span class="subtitle">Enhancing absorption and Tolerance to cadmium ions</span></div>
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   <p><strong>Cloning mnt into psb1c3 plasmid</strong><br />
   <p><strong>Cloning mnt into psb1c3 plasmid</strong><br />
     We got the mnt sequence from NCBI<a title="http://ppt.cc/Unhg" >(2)</a><a title="http://ppt.cc/feOU" >(3)</a><a title="http://ppt.cc/9-X5" >(4).</a> Next, we grew <em>Synechocystis sp. PCC 6803</em> and cloned  the mnt gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells. The plasmid we used was got  from iGEM 2012 distribution kit.<br />
     We got the mnt sequence from NCBI<a title="http://ppt.cc/Unhg" >(2)</a><a title="http://ppt.cc/feOU" >(3)</a><a title="http://ppt.cc/9-X5" >(4).</a> Next, we grew <em>Synechocystis sp. PCC 6803</em> and cloned  the mnt gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells. The plasmid we used was got  from iGEM 2012 distribution kit.<br />
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                 <li><a title="Methods & Materials" href="http://2012.igem.org/Team:NYMU-Taipei/ymic3.html">Methods & Materials</a></li>
                 <li><a title="Methods & Materials" href="http://2012.igem.org/Team:NYMU-Taipei/ymic3.html">Methods & Materials</a></li>
                 <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="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="Results & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Results &amp; References</a></li><li><a title="Further Experiments after Asia Jamboree" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq5.html">Further Experiments after Asia Jamboree</a></li>
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Latest revision as of 01:50, 27 October 2012

NYMU iGEM

Methods & Materials

Kill-switch of amoeba

Cloning zinTp into psb1c3 plasmid
We got the zinTp sequence from RegulonDB (1). Amplification was performed for 40 cycles of denaturation for 30 sec at 94 °C, annealing for 1 min at 59 °C and polymerization for 1 min at 68 °C. A PCR product containing the zinTp was cloned into plasmid psb1c3. The plasmid we used was got from iGEM 2012 distribution kit. The resulting plasmid was transformed into E. coli DH5αcompetent cells.

Characterizing of zinTp
We ligated the zinTp with GFP generator(BBa_E0840) and tested the function of zinTp. The transgenic E.coli contains zinTp+GFP, which should fluoresce when Cd2+ exist. We added different concentration of cadmium ion into LB medium and compared the fluorescence level of E.coli which contain zinTp+GFP, pTetR+GFP (BBa_I13522), and psb1c3 backbone only(wildtype control).


We grew E.coli to OD595=0.3~0.4, then added cadmium ion into micro centrifuge tubes. The E.coli was incubated at 37°C. We tested the fluorescence level of GFP. The excitation wavelength is 501nm and the emission wavelength is 511nm.


Enhancing absorption and Tolerance to cadmium ions

Cloning mnt into psb1c3 plasmid
We got the mnt sequence from NCBI(2)(3)(4). Next, we grew Synechocystis sp. PCC 6803 and cloned the mnt gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells. The plasmid we used was got from iGEM 2012 distribution kit.

Cloning smtA into psb1c3 plasmid
Firstly, we got the smtA sequence from NCBI (5). Next, we grew Synechococcus elongatus PCC 7942 and cloned the smtA gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells.


Characterizing of smtA
We cloned the smtA gene into the plasmid Ptrc-PNSII-Strep. This plasmid includes a promoter Ptrc. We compared the tolerance level between Ptrc-smtA-PNSII-Strep E.coli and E.coli that did not contain smtA(Ptrc-PNSII-Strep plasmid as a wild type control). We tested the growth curve of E.coli with different cadmium concentration.

The E.coli was grown to OD595=0.3~0.4, then we added 200λ broth into 6.cc liquid LB medium with Streptomycin 25 μg/ml. We incubated the E.coli at 37 °C.