Team:Tokyo-NoKoGen/metallothionein

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<td height=5843 rowspan=2><p class="style30">Metallothioneins (metal-binding <span class="style54">proteins</span>) and metal transporters</p>
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<p class="style6"><strong><span class="style53">1. Background</span></strong></p>
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<p class="style6">Heavy metals such as cadmium(II) and arsenic(III) used in industry and urban areas are deposited into the land and ocean. They are taken into our body through drinking water, fish and crops, which are causing serious problems against human health. To get rid of them from contaminated soil and water is a serious issue we need to solve and think about.</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/7/74/Metallothionein1.jpg" border=0 width=430 height=239 alt="metallothionein1" style="vertical-align:baseline">                          </p>
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<p class="style6">Proteins called metallothionein, which can bind to metal ions, have been reported. By using such property of metallothionein, we have decided to make a metal ion cleaning device. Our metal-cleaning system will work like this;  
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<p class="style6">1.) Make an <i>E. coli</I> that can produce metallothionein inside the cell.  
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<p class="style6">2.) Make <i>E. coli</i> synthesize transporters to take in metal ions from its surroundings, to make the metal cleaning faster and more effective.
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<p class="style6">3.) The absorbed metal ions will bind specifically to the metallothionein, which will then be collected inside the BMC (bacterial microcompartment). </p>
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<p class="style26">&nbsp;</p>
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<td height=5779 colspan=3><p class="style30">Metallothioneins (metal-binding <span class="style54">proteins</span>) and metal transporters</p>
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<p class="style56">What are metallothioneins?</p>
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<p class="style6"><strong><span class="style53">1. Background</span></strong></p>
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<p class="style6">We will focus on two metallothioneins, each paired up with transporters. Team Groningen in iGEM2009 introduced fMT (an arsenic binding metallothionein) and Glpf (arsenic transporter). We will further use and characterize their parts in our metal cleaning <i>E.coli</I> to collect arsenite. A new metallothionein that we will introduce this year in iGEM, will be SmtA (Cadmium binding metallothionein). </p>
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<p class="style6">Heavy metals such as Cd(II) and As(III) used in industry and urban are deposited into the land and ocean. They are taken into our body through drinking water, fish and crops, which are causing serious problem against human health. To get rid of them from contaminated soil and water is a serious issue we need to solve and think about.</p>
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<p class="style6">SmtA is found in <i>Synechococcus</i> sp. PCC7942 and has been reported that the cyanobacterial strain expressing SmtA reaches a higher OD550 in a cadmium containing medium. </p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/7/74/Metallothionein1.jpg" border=0 width=430 height=239 alt="metallothionein1" style="vertical-align:baseline">                          </p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/d/dd/Metallothionein2.jpg" border=0 width=506 height=255 alt="metallothionein2" style="vertical-align:baseline"></p>
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<p class="style6">Today, proteins called metallothionein that can bind to metal ions are reported. By using such property of metallothionein, we have decided to make a metal ion cleaning device. Our metal cleaning system will work like this &#8211; we will make an E.coli that can produce metallothionein inside the cell. It will also synthesize transporters to take in metal ions from its surrounding, to make the metal cleaning faster and more effective. The absorbed metal ions will bind specifically to the metallothionein, which will then be collected inside the BMC (bacterial microcompartment). </p>
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<p class="style6">The <i>E.coli</I> K-12 derived MntH (yfep) are transporters that are highly homologous to the Nramp protein family (metal ion transporters), and are known to be able to transport a variety of metal ions including Cd2+ [2]. A study has shown that MntH facilitates transport of Mn2+ in a time-, temperature-, and proton-dependent manner.</p>
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<p class="style26">&nbsp;</p>
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<p class="style6">To make metallothionein be taken into the BMC, we will fuse SmtA and fMT with PduP1-18 - a protein that is recognized and taken into pdu BMC (propanediol-utilizing BMC).</p>
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<p class="style56">What are metallothioneines?</p>
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<p class="style6">Our ideal system is to integrate SmtA, MntH, fMT and Glpf into our metal ion collecting E.coli to collect cadmium and arsenic ions. </p>
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<p class="style6">We will focus on two metallothioneins, each paired up with transporters. Team Groningen in iGEM2009 has introduced fMT (an arsenic binding metallothionein) and Glpf (arsenic transporter). We will further use and characterize their parts in our metal cleaning E.coli to collect arsenite. A new metallothionein that we will introduce this year in iGEM, will be SmtA (Cadmium binding metallothionein) and MntH (Cadmium transporter).</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/6/60/Metallothionein3.jpg" border=0 width=620 height=356 alt="metallothionein3" style="vertical-align:baseline"></p>
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<p class="style6">SmtA is found in Synechococcus sp. PCC7942 and has been reported that the cyanobacterial strain expressing SmtA reaches a higher OD550 in a cadmium containing medium. </p>
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<p class="style26">&nbsp;</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/d/dd/Metallothionein2.jpg" border=0 width=506 height=255 alt="metallothionein2" style="vertical-align:baseline"></p>
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<p class="style26">&nbsp;</p>
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<p class="style6">The E.coli K-12 derived MntH (yfep) are transporters that are highly homologous to the Nramp protein family (metal ion transporters), and are known to be able to transport a variety of metal ions including Cd2+ [2]. A study has shown that MntH facilitates transport of Mn2+ in a time-, temperature-, proton-dependent manner.</p>
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<p class="style56">2. Method</p>
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<p class="style6">To make metallothionein be taken into the BMC, we will fuse SmtA and fMT with PduP1-18 - a protein that is recognized and is taken into pdu BMC (propanediol-utilizing BMC).</p>
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<p class="style6">Our aim is to construct a vector with transporter under a constitutive promoter, and the metallothionein under a metal-sensitive promoter (Fig 4).</p>
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<p class="style6">We will integrate SmtA, MntH, fMT and Glpf into our metal ion collecting E.coli to collect cadmium and arsenic ions. </p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/1/10/Metallothionein4.jpg" border=0 width=667 height=344 alt="metallothionein4" style="vertical-align:baseline"></p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/6/60/Metallothionein3.jpg" border=0 width=620 height=356 alt="metallothionein3" style="vertical-align:baseline"></p>
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<p class="style6"><strong><span class="style53">2-1. Cloning SmtA from <i>Synechococcus</I> sp. PCC7942</span></strong></p>
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<p class="style26">&nbsp;</p>
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<p class="style6">SmtA sequence shown in red, Restriction sites shown in pink</p>
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<p class="style26">&nbsp;</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/a/a9/Metallothionein_seq1.jpg" border=0 width=406 height=98 alt="metallothioneinseq1a" style="vertical-align:baseline"> </p>
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<p class="style56">2. Method</p>
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<p class="style6">Primers to clone and add restriction sites <i>Eco</I>RI, <i>Xba</I>I and <i>Spe</I>I. </p>
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<p class="style6">Our aim is to construct a vector with transporter under a constitutive promoter, and the metallothionein under a metal-sensitive promoter as shown on the diagram.</p>
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<p class="style6">Fw primer:  AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/1/10/Metallothionein4.jpg" border=0 width=667 height=344 alt="metallothionein4" style="vertical-align:baseline"></p>
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<p class="style6">Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG</p>
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<p class="style6"><strong><span class="style53">2-1. Cloning SmtA from Synechococcus sp. PCC7942</span></strong></p>
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<p class="style6">&nbsp;</p>
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<p class="style6">SmtA sequence shown in red, Restriction sites shown in pink</p>
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<p class="style56">2-2. Cloning MntH</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/a/a9/Metallothionein_seq1.jpg" border=0 width=406 height=98 alt="metallothioneinseq1a" style="vertical-align:baseline"> </p>
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<p class="style6">MntH sequence shown in green, Restriction sites shown in pink</p>
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<p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI. </p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/2/22/Metallothionein_seq2.jpg" border=0 width=488 height=220 alt="metallothioneinseq2" style="vertical-align:baseline"></p>
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<p class="style6">Fw primer:  AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC</p>
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<p class="style6"> <img src="https://static.igem.org/mediawiki/2011/8/84/Metallothionein5.jpg" border=0 width=465 height=355 alt="metallothionein5" style="vertical-align:baseline"></p>
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<p class="style6">Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG</p>
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<p class="style6">Primers to clone and add restriction sites <i>Eco</I>RI, <i>Xba</I>I and <i>Spe</I>I.</p>
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<p class="style6">&nbsp;</p>
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<p class="style6">Fw primer:  AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC</p>
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<p class="style56">2-2. Cloning MntH</p>
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<p class="style6">Rv primer: GCTACTAGTAGGAGCACAAT</p>
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<p class="style6">MntH sequence shown in green, Restriction sites shown in pink</p>
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<p class="style6">The cloned products were cut at <i>Eco</i>RI and <i>Spe</i>I and ligated to PSB1C3 vector which was also cut at <i>Eco</i>RI and <i>Spe</i>I. </p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/2/22/Metallothionein_seq2.jpg" border=0 width=488 height=220 alt="metallothioneinseq2" style="vertical-align:baseline"></p>
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<p class="style6">&nbsp;</p>
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<p class="style6"> <img src="https://static.igem.org/mediawiki/2011/8/84/Metallothionein5.jpg" border=0 width=465 height=355 alt="metallothionein5" style="vertical-align:baseline"></p>
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<p class="style6"><strong><span class="style53">2-3. C<span class="style18">onstruct PduP1-18 fused to SmtA and fMT</span></span></strong></p>
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<p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI.</p>
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<p class="style5">We originally had PduP1-18 fused with GFP, so we carried out inverse PCR to amplify the part without GFP. We then cut the product at EcoRI and SpeI to add them to the vector containing metallothionein which were cut at EcoRI and XbaI. </p>
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<p class="style6">Fw primer:  AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC</p>
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<p class="style59">&nbsp;</p>
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<p class="style6">Rv primer: GCTACTAGTAGGAGCACAAT</p>
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<p class="style49"><strong><span class="style18">2-4. Characterize the effect of expressing SmtA and GlpF in <i>E.coli</i> cultured in Cd(II) containing medium.</span></strong></p>
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<p class="style6">The cloned products were cut at EcoRI and SpeI and ligated to PSB1C3 vector which was also cut at EcoRI and SpeI. </p>
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<p class="style49"><span class="style18"> <p class="style6"><span class="style18">LB medium with dif</span>ferent cadmium concentrations (0,100, 120, 150, 180, 210, 240, 270, 300, 400 </span><span class="style47">µ</span><span class="style18">M) was prepared. 150 </span><span class="style47">µ</span><span class="style18">L each were added to wells of a microtiter plate. WT <i>E.coli</i>, <i>E.coli</I> expressing PduP1~18-SmtA, and <i>E.coli</I> expressing PduP1~18-fMT were pre-cultured for 12 hours at 37°C. 1</span><span class="style47">µ</span><span class="style18">L of each were added to the Cd(II) containing medium(Fig.6). The change in OD595 was measured every hour and plotted against time. </p>
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<p class="style6">&nbsp;</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/9/9c/Metallothionein6.jpg" border=0 width=566 height=311 alt="metallothionein6" style="vertical-align:baseline"></p>
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<p class="style6"><strong><span class="style53">2-3. C<span class="style18">onstruct PduP1-18 fused to SmtA and fMT</span></span></strong></p>
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<p class="style6">&nbsp;</p>
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<p class="style5">We originally had PduP1-18 fused with GFP, so we carried out inverse PCR to amplify the part without GFP. We then cut the product at EcoRI and SpeI to add them to the vector containing metallothionein which were cut at EcoRI and XbaI. </p>
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<p class="style6">&nbsp;</p>
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<p class="style59">&nbsp;</p>
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<p class="style56">3. Result</p>
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<p class="style49"><strong><span class="style18">2-4. Characterize the effect of expressing SmtA and GlpF in E.coli cultured in Cd(II) containing medium.</span></strong></p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/5/58/Metallothionein7.jpg" border=0 width=613 height=792 alt="metallothioneinresults" style="vertical-align:baseline"></p>
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<p class="style49"><span class="style18">LB medium with different cadmium concentrations (0, 100, 120, 150, 180, 210, 240, 270, 300, 400 </span><span class="style47">µ</span><span class="style18">M) was prepared in a microtiter plate as shown in the diagram below, and observed the change in OD595 and compared the differences between WT E.coli, E.coli expressing PduP1~18-SmtA, and E.coli expressing PduP1~18-fMT. </span></p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/c/c8/Metallothionein8.jpg" border=0 width=531 height=355 alt="metallothionein8a" style="vertical-align:baseline"></p>
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<p class="style6"><span class="style18">We will prepare a LB medium with dif</span>ferent cadmium concentrations in a microtiter plate as shown in the diagram below, and see the change in OD660  and compare the differences between WT E.coli, E.coli expressing metallothionein, and E.coli expressing transporter. </p>
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<p class="style49"><img src="https://static.igem.org/mediawiki/2011/a/a9/Metallothionein9.jpg" border=0 width=495 height=316 alt="metallothionein9" style="vertical-align:baseline"></p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/9/9c/Metallothionein6.jpg" border=0 width=566 height=311 alt="metallothionein6" style="vertical-align:baseline"></p>
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<p class="style49"><span class="style18">As Cd(II)  concentration goes up, growth of </span><em><span class="style18">E. coli</span></em><span class="style18"> starts to slow down. At 270 </span><span class="style47">µ</span><span class="style18">M , 300 </span><span class="style47">µ</span><span class="style18">M and 400 </span><span class="style47">µ</span><span class="style18">M Cd(II) concentration, there is no difference in the OD595 between </span><em><span class="style18">E. coli </span></em><span class="style20">WT and mutant expressing </span><span class="style18">PduP1~18-fMT</span><span class="style20"> or <span class="style18">PduP1~18-</span>SmtA (Fig.7)</span><em><span class="style18">.</span></em><span class="style18"> However, at 240 </span><span class="style47">µ</span><span class="style18">M Cd(II) medium, </span><em><span class="style18">E. coli</span></em><span class="style18"> expressing PduP1~18-fMT showed a rise in its OD595 at around 6 hours. The difference becomes more significant as the Cd(II) concentration decreases, until it reaches a concentration of 120 </span><span class="style47">µ</span><span class="style18">M where the growth between metallothionein expressing </span><em><span class="style18">E. coli</span></em><span class="style18"> and the wild type becomes very similar.  Unfortunately we could not see SmtA to function as a metallothionein, as it showed a similar growth curve to the wild type </span><em><span class="style18">E. coli</span></em><span class="style18">. However, looking at the graphs showing growth curves at Cd(II) concentrations 150 </span><span class="style47">µ</span><span class="style18">M, 180 </span><span class="style47">µ</span><span class="style18">M and 210 </span><span class="style47">µ</span><span class="style18">M, cells expressing fMT shows a significant growth when compared to the growth of wild type. This result suggests that fMT bound to Cd(II) taken up by the cell, and allowed them to resist Cd(II) better than the cells without metallothionein. Our PduP1~18 fused fMT showed to maintain its function inside the <i>E. coli</I></span>.</p>
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<p class="style6">&nbsp;</p>
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<p class="style52">&nbsp;</p>
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<p class="style6">&nbsp;</p>
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<p class="style52">&nbsp;</p>
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<p class="style56">3. Result</p>
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<p class="style48"><span class="style57">4. Summary</span></p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/5/58/Metallothionein7.jpg" border=0 width=613 height=792 alt="metallothioneinresults" style="vertical-align:baseline"></p>
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<p class="style49"><span class="style18">Absorb heavy metal ions from the environment, capture them inside the cell and store them inside the BMC, which will then be collected by collecting the <i>E. coli</I>, is our metal ion collecting system using <i>E. coli</I>. The key features in our system is the use of BMC for storage and metallothioneins for capturing. Metallothioneins, proteins that can bind to metal ions we have decided to use SmtA and fMT which are known to bind to Cd(II). Having observed the difference in the growth curve of wild type <i>E. coli</I>, <i>E. coli</i> expressing SmtA, and <i>E. coli</i> expressing fMT in different LB medium of different Cd(II) concentrations, we could see the difference that <i>E. coli</i> expressing fMT could resist higher Cd(II) concentration than the wild type. Concluding from this result, fMT could bind to Cd(II) inside the cell, because it helped <i>E. coli</I> resist Cd(II). Matching our purpose of using metallothionein in <i>E. coli</i> for metal collection, we have successfully observed that fMT could still maintain its function even when fused to the PuP1~18 tag protein. The PduP1~18 fused metallothionein that captures metal ions will then become encapsulated inside the BMC and be collected.</p>
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<p class="style6"><img src="https://static.igem.org/mediawiki/2011/c/c8/Metallothionein8.jpg" border=0 width=531 height=355 alt="metallothionein8a" style="vertical-align:baseline"></p>
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<p class="style59">&nbsp;</p>
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<p class="style49"><img src="https://static.igem.org/mediawiki/2011/a/a9/Metallothionein9.jpg" border=0 width=495 height=316 alt="metallothionein9" style="vertical-align:baseline"></p>
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<p class="style49">&nbsp;</p>
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<p class="style49"><span class="style18">As Cd(II)  concentration goes up, growth of </span><em><span class="style18">E. coli</span></em><span class="style18"> starts to slow down. At 270 µM , 300 µM and 400 µM Cd(II) concentration, there is no difference in the OD595 between </span><em><span class="style18">E. coli </span></em><span class="style20">WT and mutant expressing </span><span class="style18">PduP1~18-fMT</span><span class="style20"> or <span class="style18">PduP1~18-</span>SmtA (Fig.7)</span><em><span class="style18">.</span></em><span class="style18"> However, at 240 µM Cd(II) medium, </span><em><span class="style18">E. coli</span></em><span class="style18"> expressing PduP1~18-fMT showed a rise in its OD595 at around 6 hours. The difference becomes more significant as the Cd(II) concentration decreases, until it reaches a concentration of 120 µM where the growth between metallothionein expressing </span><em><span class="style18">E. coli</span></em><span class="style18"> and the wild type becomes very similar.  Unfortunately we could not see SmtA to function as a metallothionein, as it showed a similar growth curve to the wild type </span><em><span class="style18">E. coli</span></em><span class="style18">. However, looking at the graphs showing growth curves at Cd(II) concentrations 150 µM, 180 mM and 210 mM, cells expressing fMT shows a significant growth when compared to the growth of wild type. This result suggests that fMT bound to Cd(II) taken up by the cell, and allowed them to resist Cd(II) better than the cells without metallothionein.  Our PduP1~18 fused fMT showed to maintain its function inside the <em>E. coli</em></span>.</p>
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<p class="style56">5. Reference</p>
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<p class="style88"><img src="https://static.igem.org/mediawiki/2011/e/e1/Metallothionein10.jpg" border=0 width=630 height=473 alt="metallothionein10" style="vertical-align:baseline"></p>
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<p class="style6">[1] Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene<span class="style58">.</span> <em>J Mar Biotechnol</em></p>
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<p class="style6 f-lp">[2] Makui et al. (2000) Identification of <i>Escherichia coli</I> K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter<span class="style58">.</span>  <em>Molecular Microbiology </em> </p>
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<td height=473><img src="https://static.igem.org/mediawiki/2011/6/6c/Metallothionein11.jpg" border=0 width=630 height=473 alt="metallothionein11" style="float:left"></td>
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<td height=1774 colspan=3><p class="style88 f-fp"><span class="style29">The graphs in Fig.10 and Fig. 11 show growth of </span><span class="style31">E. coli </span><span class="style29">that makes SmtA in different concentrations of Cd(II). In the previous experiment, we could not observe the tolerance of <em>E. coli</em> expressing PduP1-18-SmtA in Cd(II) containing LB medium,  but when we expressed only the SmtA inside the <em>E. coli</em>, we could see that it could tolerate the Cd(II) without the PduP1-18. This could suggest that PduP1-18 fused to SmtA changed the conformation of SmtA protein, and prevented it from binding with Cd(II).  </span></p>
 +
<p class="style88">&nbsp;</p>
 +
<p class="style88"><img src="https://static.igem.org/mediawiki/2011/3/3f/Metallothionein12.jpg" border=0 width=630 height=473 alt="metallothionein12" style="vertical-align:baseline"></p>
 +
<p class="style88">&nbsp;</p>
 +
<p class="style88"><img src="https://static.igem.org/mediawiki/2011/2/25/Metallothionein13.jpg" border=0 width=630 height=473 alt="metallothionein13" style="vertical-align:baseline"></p>
 +
<p class="style49"><span class="style29">Due to the fact that not much difference could be observed in the OD595 at 300 </span><span class="style32">m</span><span class="style29">M Cd(II) containing medium, we tried changing the promoters for expressing SmtA and PduP1-18-fMT.</span></p>
 +
<p class="style49"><span class="style29">Previously , both SmtA and PduP1-18-fMT were expressed under a relatively low constitutive promoter (BBa_J23117). This time, we changed this promoter to a high constitutive promoter (BBa_J23100) and observed the growth in 300 µM Cd(II) medium, where growth of </span><span class="style31">E. coli </span><span class="style29">could not be observed before. However, </span><span class="style31">E. coli </span><span class="style29">with a high constitutive promoter could tolerate the Cd(II) containing medium better than the </span><span class="style31">E. coli </span><span class="style29">with low constitutive promoter (Fig.12, Fig.13). This suggests that the tolerance could be due to the amount of metallothionein being expressed, with a high constitutive promoter, </span><span class="style31">E. coli </span><span class="style29">could produce more metallothioneins and therefore could resist Cd(II) better. </span></p>
 +
<p class="style49">&nbsp;</p>
 +
<p class="style49">&nbsp;</p>
 +
<p class="style49"><span class="style57"><strong>4. Summary</strong></span></p>
 +
<p class="style59"><span class="style29">Absorb heavy metal ions from the environment, capture them inside the cell and store them inside the BMC, which will then be collected by collecting the </span><span class="style31">E. coli</span><span class="style29">, is our metal ion collecting system using </span><span class="style31">E. coli</span><span class="style29">. The key features in our system is the use of BMC for storage and metallothioneins for capturing. Metallothioneins, proteins that can bind to metal ions &#8211; we have decided to use SmtA and fMT which are known to bind to Cd(II). Having observed the difference in the growth curve of wild type </span><span class="style31">E. coli</span><span class="style29">, </span><span class="style31">E. coli</span><span class="style29"> expressing SmtA, and </span><span class="style31">E. coli</span><span class="style29"> expressing PduP1-18 fused fMT in different LB medium of different Cd(II) concentrations, we could see the difference that </span><span class="style31">E. coli</span><span class="style29"> expressing PduP1-18 fused fMT could resist higher Cd(II) concentration than the wild type. Concluding from this result, fMT could bind to Cd(II) inside the cell, because it helped </span><span class="style31">E. coli</span><span class="style29"> resist Cd(II). This result is supported by our experiment of changing the promoter from Pconst(Low) to Pconst(High),  where </span><span class="style31">E. coli </span><span class="style29">expressing metallothioein under Pconst(High) could resist Cd(II) concentration better.  From the result that E. coli expressing more metallothionein could survive Cd(II), it could be deduced that metallothionein is binds with the metal ion inside the cell preventing </span><span class="style31">E. coli </span><span class="style29">from cytotoxicity. </span></p>
 +
<p class="style59"><span class="style29">Matching our purpose of using metallothionein in </span><span class="style31">E. coli</span><span class="style29"> for metal collection, we have successfully observed that fMT could still maintain its function even when fused to the PuP1~18 tag protein. The PduP1~18 fused metallothionein that captures metal ions will then become encapsulated inside the BMC and be collected.</span></p>
 +
<p class="style59"><span class="style29">We have successfully managed to observe that expressing metallothionein inside the </span><span class="style31">E. coli</span><span class="style29"> can help them tolerate Cd(II) containing medium,  and that the protein are functioning by binding with Cd(II) inside the bacterial cell and preventing cytotoxicity. </span></p>
 +
<p class="style59">&nbsp;</p>
 +
<p class="style49">&nbsp;</p>
 +
<p class="style56">5. Reference</p>
 +
<p class="style6">[1] Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene<span class="style58">.</span> <em>J Mar Biotechnol</em></p>
 +
<p class="style6 f-lp">[2] Makui et al. (2000) Identification of Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter<span class="style58">.</span>  <em>Molecular Microbiology </em> </p>
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Latest revision as of 03:11, 29 October 2011

Metallothionein v3

Tokyo-NokoGen 2011

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology

 

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Metallothioneins (metal-binding proteins) and metal transporters

1. Background

Heavy metals such as Cd(II) and As(III) used in industry and urban are deposited into the land and ocean. They are taken into our body through drinking water, fish and crops, which are causing serious problem against human health. To get rid of them from contaminated soil and water is a serious issue we need to solve and think about.

metallothionein1

Today, proteins called metallothionein that can bind to metal ions are reported. By using such property of metallothionein, we have decided to make a metal ion cleaning device. Our metal cleaning system will work like this – we will make an E.coli that can produce metallothionein inside the cell. It will also synthesize transporters to take in metal ions from its surrounding, to make the metal cleaning faster and more effective. The absorbed metal ions will bind specifically to the metallothionein, which will then be collected inside the BMC (bacterial microcompartment).

 

What are metallothioneines?

We will focus on two metallothioneins, each paired up with transporters. Team Groningen in iGEM2009 has introduced fMT (an arsenic binding metallothionein) and Glpf (arsenic transporter). We will further use and characterize their parts in our metal cleaning E.coli to collect arsenite. A new metallothionein that we will introduce this year in iGEM, will be SmtA (Cadmium binding metallothionein) and MntH (Cadmium transporter).

SmtA is found in Synechococcus sp. PCC7942 and has been reported that the cyanobacterial strain expressing SmtA reaches a higher OD550 in a cadmium containing medium.

metallothionein2

The E.coli K-12 derived MntH (yfep) are transporters that are highly homologous to the Nramp protein family (metal ion transporters), and are known to be able to transport a variety of metal ions including Cd2+ [2]. A study has shown that MntH facilitates transport of Mn2+ in a time-, temperature-, proton-dependent manner.

To make metallothionein be taken into the BMC, we will fuse SmtA and fMT with PduP1-18 - a protein that is recognized and is taken into pdu BMC (propanediol-utilizing BMC).

We will integrate SmtA, MntH, fMT and Glpf into our metal ion collecting E.coli to collect cadmium and arsenic ions.

metallothionein3

 

 

2. Method

Our aim is to construct a vector with transporter under a constitutive promoter, and the metallothionein under a metal-sensitive promoter as shown on the diagram.

metallothionein4

2-1. Cloning SmtA from Synechococcus sp. PCC7942

SmtA sequence shown in red, Restriction sites shown in pink

metallothioneinseq1a

Primers to clone and add restriction sites EcoRI, XbaI and SpeI.

Fw primer: AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC

Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG

 

2-2. Cloning MntH

MntH sequence shown in green, Restriction sites shown in pink

metallothioneinseq2

metallothionein5

Primers to clone and add restriction sites EcoRI, XbaI and SpeI.

Fw primer: AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC

Rv primer: GCTACTAGTAGGAGCACAAT

The cloned products were cut at EcoRI and SpeI and ligated to PSB1C3 vector which was also cut at EcoRI and SpeI.

 

2-3. Construct PduP1-18 fused to SmtA and fMT

We originally had PduP1-18 fused with GFP, so we carried out inverse PCR to amplify the part without GFP. We then cut the product at EcoRI and SpeI to add them to the vector containing metallothionein which were cut at EcoRI and XbaI.

 

2-4. Characterize the effect of expressing SmtA and GlpF in E.coli cultured in Cd(II) containing medium.

LB medium with different cadmium concentrations (0, 100, 120, 150, 180, 210, 240, 270, 300, 400 µM) was prepared in a microtiter plate as shown in the diagram below, and observed the change in OD595 and compared the differences between WT E.coli, E.coli expressing PduP1~18-SmtA, and E.coli expressing PduP1~18-fMT.

We will prepare a LB medium with different cadmium concentrations in a microtiter plate as shown in the diagram below, and see the change in OD660 and compare the differences between WT E.coli, E.coli expressing metallothionein, and E.coli expressing transporter.

metallothionein6

 

 

3. Result

metallothioneinresults

metallothionein8a

metallothionein9

As Cd(II) concentration goes up, growth of E. coli starts to slow down. At 270 µM , 300 µM and 400 µM Cd(II) concentration, there is no difference in the OD595 between E. coli WT and mutant expressing PduP1~18-fMT or PduP1~18-SmtA (Fig.7). However, at 240 µM Cd(II) medium, E. coli expressing PduP1~18-fMT showed a rise in its OD595 at around 6 hours. The difference becomes more significant as the Cd(II) concentration decreases, until it reaches a concentration of 120 µM where the growth between metallothionein expressing E. coli and the wild type becomes very similar. Unfortunately we could not see SmtA to function as a metallothionein, as it showed a similar growth curve to the wild type E. coli. However, looking at the graphs showing growth curves at Cd(II) concentrations 150 µM, 180 mM and 210 mM, cells expressing fMT shows a significant growth when compared to the growth of wild type. This result suggests that fMT bound to Cd(II) taken up by the cell, and allowed them to resist Cd(II) better than the cells without metallothionein. Our PduP1~18 fused fMT showed to maintain its function inside the E. coli.

metallothionein10

metallothionein11

The graphs in Fig.10 and Fig. 11 show growth of E. coli that makes SmtA in different concentrations of Cd(II). In the previous experiment, we could not observe the tolerance of E. coli expressing PduP1-18-SmtA in Cd(II) containing LB medium, but when we expressed only the SmtA inside the E. coli, we could see that it could tolerate the Cd(II) without the PduP1-18. This could suggest that PduP1-18 fused to SmtA changed the conformation of SmtA protein, and prevented it from binding with Cd(II).

 

metallothionein12

 

metallothionein13

Due to the fact that not much difference could be observed in the OD595 at 300 mM Cd(II) containing medium, we tried changing the promoters for expressing SmtA and PduP1-18-fMT.

Previously , both SmtA and PduP1-18-fMT were expressed under a relatively low constitutive promoter (BBa_J23117). This time, we changed this promoter to a high constitutive promoter (BBa_J23100) and observed the growth in 300 µM Cd(II) medium, where growth of E. coli could not be observed before. However, E. coli with a high constitutive promoter could tolerate the Cd(II) containing medium better than the E. coli with low constitutive promoter (Fig.12, Fig.13). This suggests that the tolerance could be due to the amount of metallothionein being expressed, with a high constitutive promoter, E. coli could produce more metallothioneins and therefore could resist Cd(II) better.

 

 

4. Summary

Absorb heavy metal ions from the environment, capture them inside the cell and store them inside the BMC, which will then be collected by collecting the E. coli, is our metal ion collecting system using E. coli. The key features in our system is the use of BMC for storage and metallothioneins for capturing. Metallothioneins, proteins that can bind to metal ions – we have decided to use SmtA and fMT which are known to bind to Cd(II). Having observed the difference in the growth curve of wild type E. coli, E. coli expressing SmtA, and E. coli expressing PduP1-18 fused fMT in different LB medium of different Cd(II) concentrations, we could see the difference that E. coli expressing PduP1-18 fused fMT could resist higher Cd(II) concentration than the wild type. Concluding from this result, fMT could bind to Cd(II) inside the cell, because it helped E. coli resist Cd(II). This result is supported by our experiment of changing the promoter from Pconst(Low) to Pconst(High), where E. coli expressing metallothioein under Pconst(High) could resist Cd(II) concentration better. From the result that E. coli expressing more metallothionein could survive Cd(II), it could be deduced that metallothionein is binds with the metal ion inside the cell preventing E. coli from cytotoxicity.

Matching our purpose of using metallothionein in E. coli for metal collection, we have successfully observed that fMT could still maintain its function even when fused to the PuP1~18 tag protein. The PduP1~18 fused metallothionein that captures metal ions will then become encapsulated inside the BMC and be collected.

We have successfully managed to observe that expressing metallothionein inside the E. coli can help them tolerate Cd(II) containing medium, and that the protein are functioning by binding with Cd(II) inside the bacterial cell and preventing cytotoxicity.

 

 

5. Reference

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

[2] Makui et al. (2000) Identification of Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter. Molecular Microbiology