Team:NYMU-Taipei/ymic3.html

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<div class="title">Experiment Design</div>
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<div class="title">Methods & Materials</div>
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   <p>        <strong><em>Into  the cell
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      <span class="subtitle">Kill-switch of amoeba</span></div>
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          By  cloning LLO and invasin into the E.coli, the bacteria can get into the amoeba  and live inside it. Invasin helps the cell to be endocytosed by the host. Then Listeriolysin O makes the cell escaped from phagosomes in the  cytoplasm of the host. <br />
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   <p><strong>Cloning zinTp into psb1c3 plasmid</strong><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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<strong>Characterizing of zinTp</strong><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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    <p align="center"><img src="../../../Users/jaymes/Desktop/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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             <strong><em>Cadmium accumulation</em></strong><br />
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        To  enhanced E.coli&rsquo;s ability of gathering cadmium ions, we clone smtA and mnt gene into the E.coli. SmtA is a cadmium binding metallothionein, which can bind to cadmium ions. Mnt is an ion transporter that  pumps cadmium ions inside the bacteria.</p>
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     <p align="center"><img src="https://static.igem.org/mediawiki/2012/6/6b/Ymic3.png" alt="" width="471" height="292" /><br />
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     <span class="subtitle">Enhancing absorption and Tolerance to cadmium ions</span></div>
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      The main idea of how E.coli and Dictyostelium discoideum system works.<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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    </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="../../../Users/jaymes/Desktop/ymic11.png" alt="" width="558" height="357" /><br />
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<strong>Characterizing of smtA<br />
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           ---<strong><em>smtA</em></strong><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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  <strong>        </strong>Metallothionein (MT) is a family  of cysteine-rich, low molecular weight (MW ranging from 500 to 14000 Da)  proteins. MTs have the capacity to bind both physiological (such as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver, arsenic)  heavy metals through the thiol group of its cysteine residues<a title="Sigel, A.; Sigel, H.; Sigel, R.K.O., ed. (2009). Metallothioneins and Related Chelators. Metal Ions in Life Sciences. 5. Cambridge: RSC Publishing." >(1)</a>.<br />
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  Synechococcus PCC. 7942  gene smtA encodes the protein designated to be a metallothionein (MT). While expressing this gene in E.coli, it can increase cadmium ion tolerance of E.coli and so does the accumulation of cadmium ion<a title="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" >(2)</a><a title="Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene. J Mar Biotechnol" >(3)</a>.<div class=out style='text-align:center'>
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    <p align="center"><img src="https://static.igem.org/mediawiki/2012/1/1f/Ymic4.png" alt="" width="359" height="223" /><br />
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          ---<strong>mnt</strong><br />
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        Mnt gene is the Mg2+/Cd2+ transporter which belongs to ABC transporters system. The ATP-binding  cassette (ABC) transporters form one of the largest known protein families, and  are widespread in bacteria, archaea, and eukaryotes. They couple ATP hydrolysis to active transport. The structure of a prokaryotic ABC transporter usually  consists of three components; typically two integral membrane proteins each  having six transmembrane segments, two peripheral proteins that bind and  hydrolyze ATP, and a periplasmic (or lipoprotein) substrate-binding protein.  Many of the genes for the three components form operons as in fact observed in  many bacterial and archaeal genomes.<a title="http://ppt.cc/kBfk" >(4)</a><br />
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     <p align="center"><img src="https://static.igem.org/mediawiki/2012/1/1f/Ymic5.png" alt="" width="325" height="362" /></p>
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     <p align="center"><img src="../../../Users/jaymes/Desktop/ymic12.png" alt="" width="395" height="492" /></p>
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    <p>Cloning mntA gene into E.coli can increase  Cd2+ uptake.    </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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      <span class="subtitle">Biosafety Consideration</span></div>
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    <p><strong>---</strong> <strong>Precaution  against </strong><strong>Horizontal gene transfer – Living inside of amoeba</strong><br />
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      Horizontal gene transfer(HGT) is a  serious issue concerning the release of genetically modified organisms (GMOs)  into the environment. To prevent HGT, we must make sure our genetic modified  E.coli will never join their wild type relations. Our solution is to put the  engineered E.coli into amoeba, so the E.coli cannot contact the environment  directly. Whenever wild type bacteria break into the amoeba, they will be  digested.<br /><div class=out style='text-align:center'>
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    <p align="center"><img src="https://static.igem.org/mediawiki/2012/7/76/Ymic6.png" alt="" width="382" height="246" /></p>
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    --- Precaution  against becoming invasive species - Kill switch of Amoeba</strong><br />
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    To make sure our  genetic modified organisms will stay in the area where we want them to clean up  cadmium ions, we also build a kill-switch that will switch on when there is no  cadmium ion in the soil. The kill-switch has two man-made operons. The first  one includes zinTp(previous name pYodA), this promoter activity is induce by  cadmium ion; and its constructive gene is lacI. The other one includes BBa_R0010,  the promoter whose activity is inhibited by the protein lacI; and the  constructive gene DdaifA and HlyA(BBa_K223054). DdaifA is a protein that cause  the apoptosis of Dictyostelium discoideum cell. HlyA is a tag that stick after  the target protein. With this tag, E.coli will secrete the target protein. When  cadmium ions exist, zinTp works and lacI is generated, so the expression of  aifA and HlyA is repressed. If there is no cadmium ions, BBa_R0010 will  activate the expression of aifA and HlyA, so the protein aifA will be secrete  right into the cytoplasm of amoeba, causing amoeba apoptosis.<br />
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     <p align="center"><img src="https://static.igem.org/mediawiki/2012/4/47/Ymic7.png" width="500" height="385" /></p>
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    zinTp</strong>
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  <p>    ZinTp (pYodA) is a promoter which expresses  the downstream gene in the presence of cadmium ion. The activity of this  promoter is specific affect by cadmium ion and won&rsquo;t be induced by other ions  like zinc, copper, cobalt, and nickel<a title="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" >(5)</a>. <div class=out style='text-align:center'>
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    <p align="center"><img src="https://static.igem.org/mediawiki/2012/a/a6/Ymic8.png" alt="" width="191" height="201" /></p>
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    <p align="left">Strain AL6 (λФ PyodA–lacZ) was grown aerobically in LB medium at  37°C in presence (●) or absence (○) of cadmium (136±5μM)      <em>β</em>-Galactosidase activity in the
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      presence (■) or absence (□) of tested  agents was also measured.</p>
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      DdaifA </strong>
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  <p>    Apoptosis-inducing  factor (AIF) is involved in a caspase-independent cell death pathway. <em>Dictyostelium discoideum</em> has a homolog  of mammalian AIF, which could cause nuclei breakdown, leading the cell to the  apoptosis program<a title="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" >(6)</a>.<br /><div class=out style='text-align:center'>
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    <p align="center"><img src="https://static.igem.org/mediawiki/2012/f/ff/Ymic9.png" alt="" width="202" height="225" /></p>
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    <p align="left">DdAIF induces damage of mammalian and Dictyostelium nuclei in a cell-free system. (E) shows the quantification of damaged nuclei induced by cytoplasmic extracts.
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Damaged nuclei were assessed by the disappearance of the nucleoli. One hundred nuclei were counted in each condition. Histograms are the mean of three independent experiments. Nu, nucleolus; Dnu, dying cells nucleolus. Dictyostelium cells (control CE), Dictyostelium cells treated with PPIX to cause cell death (dying cells CE). Cytoplasmic extracts of dying cells were also immunodepleted with anti-DdAIF (dying cells CE -AIF) or with nonimmune serum (dying cells CE NIS)
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Revision as of 03:45, 25 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.

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