Team:NYMU-Taipei/ymic4.html

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<div class="title">Experiment Design</div>
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<div class="title">Results & Discussion</div>
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   <p>        <strong><em>Into  the cell
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   <p><span class="subtitle">Cloning zinTp into pSB1C3 plasmid</span></p>
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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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            <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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      The main idea of how E.coli and Dictyostelium discoideum system works.<br />
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           ---<strong><em>smtA</em></strong><br />
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   <p align="center"><img src="../../../Users/jaymes/Desktop/ymic13.png" width="600" height="220" /><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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We got the right plasmid which contained zinTp<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><span class="subtitle">Characterizing of zinTp</span></p>
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    <p>Cloning mntA gene into E.coli can increase  Cd2+ uptake.    </p>
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     <div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic14.png" width="600" height="306" /><br />
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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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     <p>In this fig we could  see that before 60 min, the fluorescence level of zinTp+GFP steadily increased,  while BBa_I13522 (positive control) remained stable around 35000 au as time  gone by. At 60 min, zinTp reached its maximum transcription rate so the  fluorescence level stop increasing around 55000 au.</p><div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic16.png" alt="" width="494" height="306" /><br />
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       <strong>Fluorescence level at 15mins after Cd2+  added </strong><br />
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    <p>This fig shows the fluorescence level of  different cadmium concentration. We can see that BBa_I13522 remained stable  from different cadmium concentration, while the fluorescence level of zinTp+GFP  increased as the cadmium concentration increased. We could also tell that the  transcription rate of zinTp is fairly low when there is no cadmium ion. <br />
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       DdaifA </strong>
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   <p><span class="subtitle">Cloning mnt into pSB1C3 plasmid</span><br />
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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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      </div><div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic17.png" width="630" height="243" /><br />
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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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      Mnt gene was successfully cloned into pSB1C3 plasmid<br />
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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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        <p><span class="subtitle">Cloning mnt into pSB1C3 plasmid</span><br />
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        </p>
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      <div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic18.png" width="600" height="331" /><br />
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      smtA gene was successfully cloned into pSB1C3 plasmid<br />
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      <br /><div align="left">
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        <p><span class="subtitle">Characterizing of smtA</span><br />
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        </p>
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      </div><div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic19.png" alt="" width="508" height="342" /><br />
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        <strong>Cadmium 0/200/500μM Medium</strong><br />
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      <p>This fig shows that smtA enhances the tolerance  level to cadmium ion. In wild type E.coli, OD595 differ from between  0/200/500μM cadmium concentration. E.coli hardly grow in 500μM cadmium medium. However, the E.coli which can express smtA seem to show  no significant different between 0 &amp; 200μM cadmium medium.</p>
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      <div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic20.png" alt="" width="463" height="282" /><br />
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        <strong>8 hours after cadmium added</strong><br />
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      <p>OD of WT E.coli  decrease when cadmium concentration increase. The E.coli which can express smtA  seem to have stronger resistance to cadmium toxicity and the E.coli  concentration show no significant different from 0 to 200μM cadmium medium.</p>
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<div class="out" style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymic21.png" alt="" width="386" height="291" /><br />
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Obviously, the E.coli  which could express smtA growth better than wild type in 200μM cadmium medium. <br />
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Revision as of 01:15, 26 October 2012

NYMU iGEM

Results & Discussion

Cloning zinTp into pSB1C3 plasmid


We got the right plasmid which contained zinTp

Characterizing of zinTp





In this fig we could see that before 60 min, the fluorescence level of zinTp+GFP steadily increased, while BBa_I13522 (positive control) remained stable around 35000 au as time gone by. At 60 min, zinTp reached its maximum transcription rate so the fluorescence level stop increasing around 55000 au.


Fluorescence level at 15mins after Cd2+  added

This fig shows the fluorescence level of different cadmium concentration. We can see that BBa_I13522 remained stable from different cadmium concentration, while the fluorescence level of zinTp+GFP increased as the cadmium concentration increased. We could also tell that the transcription rate of zinTp is fairly low when there is no cadmium ion.


Cloning mnt into pSB1C3 plasmid


Mnt gene was successfully cloned into pSB1C3 plasmid


Cloning mnt into pSB1C3 plasmid


smtA gene was successfully cloned into pSB1C3 plasmid

Characterizing of smtA


Cadmium 0/200/500μM Medium

This fig shows that smtA enhances the tolerance level to cadmium ion. In wild type E.coli, OD595 differ from between 0/200/500μM cadmium concentration. E.coli hardly grow in 500μM cadmium medium. However, the E.coli which can express smtA seem to show no significant different between 0 & 200μM cadmium medium.


8 hours after cadmium added

OD of WT E.coli decrease when cadmium concentration increase. The E.coli which can express smtA seem to have stronger resistance to cadmium toxicity and the E.coli concentration show no significant different from 0 to 200μM cadmium medium.



Obviously, the E.coli which could express smtA growth better than wild type in 200μM cadmium medium.

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