Team:NYMU-Taipei/ymic5.html

From 2012.igem.org

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<div class="title">Methods & Materials</div>
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<div class="title">Conclusion & References</div>
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    <li>We have successfully  cloned zinTp into pSB1C3</li>
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    <li>We have successfully  cloned zinTp-GFP into pSB1C3</li>
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    <li>We have characterized  zinTp function. The expression of downstream gene will increase when added  cadmium ion into LB medium, and the maximum transcription rate occurs around 60  min after added 200μM cadmium.</li>
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    <li>We have successfully  cloned mnt into pSB1C3</li>
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    <li>We have successfully  cloned smtA into pSB1C3</li>
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    <li>We have characterized  smtA function. When expressing smtA, it enhances E.coli tolerance to cadmium.<br />
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      <span class="subtitle">Kill-switch of amoeba</span><br />
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    <span class="subtitle">References</span></div>
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      Sigel, A.; Sigel, H.; Sigel, R.K.O., ed. (2009). <em>Metallothioneins and Related Chelators</em>. Metal Ions in Life  Sciences. Cambridge: RSC Publishing<br />
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  <p><strong>Cloning zinTp into psb1c3 plasmid</strong><br />
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    Jianguo Shi,  William P. Lindsay, James W. Huckle, Andrew P. Morby and Nigel J. Robinson. Cyanobacterial  metallothionein gene expressed in Escherichia coli Metal-binding properties of the expressed protein. FEB.5 11081 Volume 303, number 2,3, 159-163<br />
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    We got the zinTp sequence from RegulonDB <a title="http://ppt.cc/SS5N" >(1)</a>. 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.
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    Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene. <em>J Mar Biotechnol</em> <br />
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<strong>Characterizing of zinTp</strong><br />
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     ZHIQI HAO,1  SHAOLIN CHEN,1 AND DAVID B. WILSON1,2,   Cloning, Expression, and Characterization of Cadmium and Manganese  Uptake Genes from <em>Lactobacillus plantarum,</em> APPLIED AND ENVIRONMENTAL  MICROBIOLOGY,Nov. 1999, p.4746–4752 Vol. 65, No. 11 <br />
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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). </p>
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     A. Paskarova, P. Ferianc, J. Kormanec, D. Homerova, A. Farewell, T. Nystrom. Regulation of yodA  encoding a novel cadmium-induced protein in Escherichia coli, Microbiology (2002), 148, 3801–3811 <br />
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     <p align="center"><img src="http://2012.igem.org/wiki/images/f/f0/Ymic10.png" alt="" width="495" height="311" /><br />
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      <p align="left">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. <br />
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     <span class="subtitle">Enhancing absorption and Tolerance to cadmium ions</span><br />
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  <p><strong>Cloning mnt into psb1c3 plasmid</strong><br />
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     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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     <strong></strong><strong>Cloning smtA into psb1c3 plasmid<br />
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     Damien Arnoult, Ire`ne Tatischeff, Je´rome Estaquier, Mathilde Girard, Franck Sureau, Jean Pierre Tissier, Alain Grodet, Marc Dellinger, Francois Traincard,Axel Kahn,Jean-Claude Ameisen, and Patrice Xavier Petit. &ldquo;On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor during <em>Dictyostelium  discoideum</em> Cell Death&rdquo;, Molecular Biology of the Cell Vol. 12, 3016–3030,  October 2001<br />
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    </strong>Firstly, we got the smtA sequence from NCBI <a title="http://ppt.cc/t9kR" >(5)</a>. Next, we grew <em>Synechococcus elongatus</em> PCC 7942 and cloned the smtA gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells.  
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    <p align="center"><img src="http://2012.igem.org/wiki/images/f/ff/Ymic11.png" alt="" width="558" height="357" /><br />
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<strong>Characterizing of smtA<br />
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</strong>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.<br />
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    <p align="center"><img src="http://2012.igem.org/wiki/images/4/4e/Ymic12.png" alt="" width="395" height="492" /></p>
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    <p align="left">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.</p>
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Revision as of 10:40, 25 October 2012

NYMU iGEM

Conclusion & References

  1. We have successfully cloned zinTp into pSB1C3
  2. We have successfully cloned zinTp-GFP into pSB1C3
  3. We have characterized zinTp function. The expression of downstream gene will increase when added cadmium ion into LB medium, and the maximum transcription rate occurs around 60 min after added 200μM cadmium.
  4. We have successfully cloned mnt into pSB1C3
  5. We have successfully cloned smtA into pSB1C3
  6. We have characterized smtA function. When expressing smtA, it enhances E.coli tolerance to cadmium.
References

Sigel, A.; Sigel, H.; Sigel, R.K.O., ed. (2009). Metallothioneins and Related Chelators. Metal Ions in Life Sciences. Cambridge: RSC Publishing

Jianguo Shi, William P. Lindsay, James W. Huckle, Andrew P. Morby and Nigel J. Robinson. Cyanobacterial metallothionein gene expressed in Escherichia coli Metal-binding properties of the expressed protein. FEB.5 11081 Volume 303, number 2,3, 159-163

Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene. J Mar Biotechnol

ZHIQI HAO,1 SHAOLIN CHEN,1 AND DAVID B. WILSON1,2,  Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus plantarum, APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Nov. 1999, p.4746–4752 Vol. 65, No. 11

A. Paskarova, P. Ferianc, J. Kormanec, D. Homerova, A. Farewell, T. Nystrom. Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli, Microbiology (2002), 148, 3801–3811

Damien Arnoult, Ire`ne Tatischeff, Je´rome Estaquier, Mathilde Girard, Franck Sureau, Jean Pierre Tissier, Alain Grodet, Marc Dellinger, Francois Traincard,Axel Kahn,Jean-Claude Ameisen, and Patrice Xavier Petit. “On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor during Dictyostelium discoideum Cell Death”, Molecular Biology of the Cell Vol. 12, 3016–3030, October 2001