Team:NYMU-Taipei/ymic2.html

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<div class="title">Cd+2 Collector</div>
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<div class="title">Experiment Design</div>
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   <p><span class="subtitle">Overview</span></p>
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   <p>        <strong><em>Into  the cell
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  </em></strong><br />
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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>We&rsquo;ve built the endosymbiotic system to clean up cadmium  contamination. The engineered organism can collect cadmium ion in the soil and can be easily gather together; moreover, the device won&rsquo;t become another pollution source to our environment.<br />
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  <br />
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     Cadmium contamination is a serious problem in many countries; cadmium can cause respiratory  system damage, renal failure, bones softening, and, maybe the most infamous case in Asia, Itai-itai disease. The rice absorbed the cadmium in the soil and  the cadmium accumulated in the people eating contaminated rice. The aim of our  project is to build up a system that can collect Cadmium ion in the soil and can be easily gather together; besides, we don&rsquo;t want our device become another  pollution source to our environment. We found that the endosymbiotic system between the soil-living amoeba <em>Dictyostelium  discoideum </em>and <em>E.coli </em>would be  our favorite solution.<br />
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             <strong><em>Cadmium accumulation</em></strong><br />
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  </p>
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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="http://2012.igem.org/wiki/images/0/09/Ymic1.gif" alt="" /><br />
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     <p align="center"><img src="../../../Users/jaymes/Desktop/ymic3.png" alt="" width="471" height="292" /><br />
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       Cadmium ions were absorbed by amoeba then  accumulated inside of E.coli<br />
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       The main idea of how E.coli and Dictyostelium discoideum system works.<br />
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  <p> <br />
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           ---<strong><em>smtA</em></strong><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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   <p><span class="subtitle">Why <em>Dictyostelium discoideum</em></span></p>
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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="../../../Users/jaymes/Desktop/ymic4.png" alt="" width="359" height="223" /><br />
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    </p>
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<em>Dictyostelium discoideum </em>is a soil-living amoeba, so it is a good choice to collect cadmium ion in soil. In addition, the amoeba naturally has endosymbiont inside the cell. The E.coli living in amoeba accumulates cadmium ions, just like an organelle, the E.coli acts as a compartment preventing its host from cadmium poisoning. Furthermore, keeping the E.coli inside the amoeba avoid unexpectedly gene exchange between engineered E.coli and other wild types bacteria.  Moreover, Dictyostelium discoideum chemotaxis is well-studied and can be easily gather by adding cAMP.<br />
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  <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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    <br /><div class=out style='text-align:center'>
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    <p align="center"><img src="../../../Users/jaymes/Desktop/ymic5.png" alt="" width="325" height="362" /></p>
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    <p>Cloning mntA gene into E.coli can increase  Cd2+ uptake.    </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 />
 +
      Horizontal gene transfer(HGT) is 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="../../../Users/jaymes/Desktop/ymic6.png" alt="" width="382" height="246" /></p>
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  </div>
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  <p><strong><br />
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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="../../../Users/jaymes/Desktop/ymic7.png" width="500" height="385" /></p>
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     <p align="center"><img src="http://2012.igem.org/wiki/images/f/fc/Ymic2.gif" alt="" /><br />
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  <ol>
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    <strong><br />
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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="../../../Users/jaymes/Desktop/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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    <ol>
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      <strong> <br />
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      <br />
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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="../../../Users/jaymes/Desktop/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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      <br />
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      <br />
     </p>
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     <p>Single cell amoeba <em>Dictyostelium discoideum</em> gathered  when cAMP was added.<br />
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Revision as of 03:23, 25 October 2012

NYMU iGEM

Experiment Design

        Into the cell
        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.

        Cadmium accumulation
        To enhanced E.coli’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.


The main idea of how E.coli and Dictyostelium discoideum system works.

 
       ---smtA
        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(1).
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(2)(3).



        ---mnt
        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.(4)

Cloning mntA gene into E.coli can increase Cd2+ uptake.




Biosafety Consideration

--- Precaution against Horizontal gene transfer – Living inside of amoeba
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.


--- Precaution against becoming invasive species - Kill switch of Amoeba

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.


    zinTp

    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’t be induced by other ions like zinc, copper, cobalt, and nickel(5).

Strain AL6 (λФ PyodA–lacZ) was grown aerobically in LB medium at 37°C in presence (●) or absence (○) of cadmium (136±5μM) β-Galactosidase activity in the presence (■) or absence (□) of tested agents was also measured.



    DdaifA

    Apoptosis-inducing factor (AIF) is involved in a caspase-independent cell death pathway. Dictyostelium discoideum has a homolog of mammalian AIF, which could cause nuclei breakdown, leading the cell to the apoptosis program(6).

DdAIF induces damage of mammalian and Dictyostelium nuclei in a cell-free system. (E) shows the quantification of damaged nuclei induced by cytoplasmic extracts. 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)