Team:Tokyo-NoKoGen/photocontrol

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<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Project"><span class="style18" style="text-decoration:underline">Project: EcoLion</span></a></span></strong></p>
<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Project"><span class="style18" style="text-decoration:underline">Project: EcoLion</span></a></span></strong></p>
<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Parts"><span class="style18" style="text-decoration:underline">BioBricks</span></a></span></strong></p>
<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Parts"><span class="style18" style="text-decoration:underline">BioBricks</span></a></span></strong></p>
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<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/notebook"><span class="style18" style="text-decoration:underline">Notebook</span></a></span></strong></p>
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<p class="style35"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/notebook"><span class="style18" style="text-decoration:underline"><strong>Notebook</strong></span></a></p>
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<p class="style3"><strong><span class="style14"><a href="Protocols"><span class="style18" style="text-decoration:underline">Protocols</span></a></span></strong></p>
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<p class="style3"><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/protocols"><span class="style18" style="text-decoration:underline"><strong>Protocols</strong></span></a></span></p>
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<p class="style3"><strong><span class="style14"><a href="attribution"><span class="style18" style="text-decoration:underline">Attribution</span></a></span></strong></p>
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<p class="style3"><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/attribution"><span class="style18" style="text-decoration:underline"><strong>Attribution</strong></span></a></span></p>
<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/safety"><span class="style18" style="text-decoration:underline">Safety</span></a></span></strong></p>
<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/safety"><span class="style18" style="text-decoration:underline">Safety</span></a></span></strong></p>
<p class="style3 f-lp"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/sponsors"><span class="style18" style="text-decoration:underline">Sponsors</span></a></span></strong></p>
<p class="style3 f-lp"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/sponsors"><span class="style18" style="text-decoration:underline">Sponsors</span></a></span></strong></p>
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<td height=676 colspan=2><p class="style63">Photocontrol</p>
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<td height=2313 colspan=2><p class="style30">Metallothioneins (metal-binding <span class="style54">proteins</span>) and metal transporters</p>
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<p class="style41"><span class="style62">- phototaxis, aggregation, and lysis</span></p>
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<p class="style6"><strong><span class="style53">1. Background</span></strong></p>
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<p class="style43">&nbsp;</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="style64">Introduction</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="style43">After having collected heavy metals using transporters and metallothioneins, and storing them inside the BMC, comes the last part of the project &#8211; how do we collect <em>E. coli</em> that has absorbed the toxic compounds? The whole procedure will be done in a large scale, and so we should think of an efficient and a convenient way for collecting the <em>E. coli</em>. The word efficiency is often used for robots and machines. They make our life convenient, by making us do less work. It would be fun if we can make <em>E. coli</em> become convenient for us, and can make them easy to control and make them move like robots and machines. An input used for robots and machines are electricity, but what can we use for <em>E. coli</em> as an input for movements? Today, there are several research and reports on <em>E. coli </em>reacting to light, a word known as phototaxis. </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="style43">Phototaxis is a property of <em>E. coli,</em> that allows movement of <em>E. coli</em> reacting to light. Last year we have found a few reports that have tried to engineer <em>E. coli</em> to add phototaxis, research based on Halophilic archaea [1, 2]. We have referred to the reported papers and tried creating a phototaxis device for collecting <em>E. col</em>i in an efficient way. By using light, it is possible to control the movement of <em>E. coli</em> and we can use it for collection in our system</p>
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<p class="style26">&nbsp;</p>
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<p class="style43"><img src="https://static.igem.org/mediawiki/2011/4/4a/Phototaxisfig1.jpg" border=0 width=256 height=293 alt="phototaxisfig1" style="vertical-align:baseline"></p>
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<p class="style56">What are metallothioneines?</p>
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<p class="style43">Aggregation, is another possible option for collecting E. coli. Inducing E. coli to produce aggregation proteins Antigen43, we can make the E. coli aggregate and collect them at once as a cluster (Fig.2).</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">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="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">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="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">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/2011/6/60/Metallothionein3.jpg" border=0 width=620 height=356 alt="metallothionein3" style="vertical-align:baseline"></p>
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<p class="style26">&nbsp;</p>
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<p class="style26">&nbsp;</p>
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<p class="style56">2. Method</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"><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"><strong><span class="style53">2-1. Cloning SmtA from Synechococcus sp. PCC7942</span></strong></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="style6"><img src="Resources/metallothioneinsa.jpeg" border=0 width=488 height=220 alt="metallothioneinseq2" style="vertical-align:baseline"> </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">Fw primer:  AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC</p>
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<p class="style6">Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG</p>
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<p class="style6">&nbsp;</p>
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<td height=207><img src="https://static.igem.org/mediawiki/2011/9/91/Phototaxisfig2.jpg" border=0 width=374 height=207 alt="phototaxisfig2" style="float:left"></td>
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<td height=4755 colspan=2><p class="style43 f-fp">What we need to put in mind as a consideration is &#8211; what do we do when we fail to collect some of the E. coli? To solve the problem, we have decided to introduce lysis genes in to the E. coli. The whole procedure for collecting heavy metals from contaminated water can be done at night in the dark. The collection of heavy metals and the E. coli should be finished by the time the sun comes out. Lysis genes will be turned on by red light, leading to the death of E. coli that has leaked out from phototaxis or aggregation (Fig.3). By introducing lysis as a consideration for biosafety, we can avoid biohazard.</p>
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<td height=3157 colspan=2><p class="style56 f-fp">2-2. Cloning MntH</p>
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<p class="style43">&nbsp;</p>
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<p class="style6">MntH sequence shown in green</p>
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<p class="style43"><img src="https://static.igem.org/mediawiki/2011/0/0e/Phototaxisfig3.jpg" border=0 width=416 height=271 alt="phototaxisfig3" style="vertical-align:baseline"></p>
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<p class="style6">Restriction sites shown in pink</p>
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<p class="style59">&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="style59">&nbsp;</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="style61">1. Phototaxis</p>
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<p class="style6">Fw primer: AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC</p>
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<p class="style55"><span class="style53">1-1. Construct</span></p>
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<p class="style6">Rv primer: GCTACTAGTAGGAGCACAAT</p>
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<p class="style59"><img src="https://static.igem.org/mediawiki/2011/4/44/Phototaxisfig4.jpg" border=0 width=435 height=119 alt="phototaxisfig4" style="vertical-align:baseline"></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="style59">&nbsp;</p>
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<p class="style6">&nbsp;</p>
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<p class="style59"><strong><span class="style53">1-2. How it works</span></strong></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="style59"><img src="https://static.igem.org/mediawiki/2011/e/e5/Phototaxisfig5.jpg" border=0 width=404 height=333 alt="phototaxisfig5" style="vertical-align:baseline"></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="style59">When light is shone, CheA phosphorylates itself, which then phosphorylates CheY. CheY then changes the movement of flagellar, and makes phototaxis happen. In the dark the E. coli swims straight, but when there is light, E. coli starts tumbling and as a result moves as if it is moving away from the light.</p>
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<p class="style59">&nbsp;</p>
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<p class="style61">1-3. Evaluation</p>
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<p class="style59">We expressed phototaxis device (BBa_K317028) in E.coli DH5&#945; and evaluated its function. The E. coli was pre-cultured in 3 mL LB medium containing Chloramphenicol at 37oC until the OD660 reached 0.5. 1 mL of the pre-culture was removed into a tube, where all-trans retinal (f.c. 2 &#956;M) was added. The tubes were wrapped in aluminium foil to avoid light, and were cultured by shaking at 37 oC for 2 hours. 10 &#956;L of pre-cultured solution was plotted onto semisolid medium of 0.5% agar and 2 &#956;M all-trans retinal, and incubated overnight under light (fluorescent light) or dark (wrapped in aluminium foil) conditions. As a result, we expect to see that the size of the colony will not change under light condition because the cells will not move, but under dark condition, we expect to see that the colonies grow larger because of the induced movement.</p>
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<p class="style59">&nbsp;</p>
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<p class="style59">&nbsp;</p>
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<p class="style61">2. Aggregation</p>
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<p class="style61">2.-1 Construct </p>
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<p class="style55"><img src="https://static.igem.org/mediawiki/2011/a/a3/Phototaxisfig6.jpg" border=0 width=355 height=116 alt="phototaxisfig6" style="vertical-align:baseline"></p>
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<p class="style55">&nbsp;</p>
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<p class="style55"><span class="style53">2</span><span class="style45">-</span><span class="style53">2. How it works</span></p>
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<p class="style59"><img src="https://static.igem.org/mediawiki/2011/2/25/Phototaxisfig7.jpg" border=0 width=458 height=334 alt="phototaxisfig7" style="vertical-align:baseline"></p>
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<p class="style59">&nbsp;</p>
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<p class="style59"><strong><span class="style53">2-3. Evaluation</span></strong></p>
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<p class="style59">The E. coli cells containing Antigen43 gene was pre-cultured in 3 mL LB medium at 37 oC for 12 hours. After adjusting its OD595, they were pre-cultured solution were suspended into 100 mL LB medium and cultured at 37 oC. Its OD595 was measured every 1 hour until the OD595 reached 2.5, and 3 mL of it was put into a test tube for IPTG induction of Antigen43 expression. After 2 hours of culturing at 37 oC, OD595 of the liquid culture 1cm from the surface was measured and plotted against a curve.</p>
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<p class="style55">&nbsp;</p>
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<p class="style55">&nbsp;</p>
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<p class="style61">3. Lysis</p>
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<p class="style55"><span class="style53">3</span><span class="style45">-</span><span class="style53">1. Construct </span></p>
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<p class="style59"><img src="https://static.igem.org/mediawiki/2011/6/6f/Phototaxisfig8.jpg" border=0 width=369 height=187 alt="phototaxisfig8" style="vertical-align:baseline"></p>
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<p class="style59">&nbsp;</p>
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<p class="style55"><span class="style53">3</span><span class="style45">-</span><span class="style53">2. How it works</span></p>
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<p class="style59"><img src="https://static.igem.org/mediawiki/2011/e/ea/Phototaxisfig9.jpg" border=0 width=458 height=241 alt="phototaxisfig9" style="vertical-align:baseline"></p>
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<p class="style59">&nbsp;</p>
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<p class="style59"><strong><span class="style53">3-3. Evaluation</span></strong></p>
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<p class="style59">Lysis genes with and without antiholin was evaluated to see any leakage of lysis gene expression. An E. coli containing an empty vector as a control, an E. coli containing Lysis genes with antiholin, and an E. coli containing Lysis genes without antiholin were cultured at 37 oC and OD660 was measured every 30min. to 1 hour.</p>
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<p class="style59">To find out if IPTG expression of lysis gene work, using the vectors containing holin and endolysin expressed under lac promoter, and antiholin expressed under a constitutive promoter, we transformed E. coli DH5&#945;. The obtained colony pre-cultured overnight in 2 mL LB and 1 mL of it was cultured in 100 mL LB at 37&#8451;. At OD660, IPTG (f.c. 3 mM) was added to induce the expression of Lysis genes. </p>
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<p class="style55">&nbsp;</p>
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<p class="style55">&nbsp;</p>
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<p class="style61">4. Results&amp;Discussion</p>
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<p class="style61">4-1. Phototaxis</p>
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<p class="style59">&nbsp;</p>
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<p class="style59">&nbsp;</p>
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<p class="style59">&nbsp;</p>
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<p class="style59">&nbsp;</p>
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<p class="style55"><span class="style53">4-2. Aggregation</span></p>
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<p class="style55"><img src="Resources/phototaxisfig10.jpeg" border=0 width=326 height=215 alt="phototaxisfig10" style="vertical-align:baseline"></p>
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<p class="style59">This is the result of antigen43 expression (fig.7).</p>
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<p class="style59">The red and orange line represents growth curve of cells containing Antigen43 under PLlacO1, and the green and the blue lines represents growth curve of cells containing no Antigen43 gene. There is a significance difference between cells with and without the aggregation proteins, as the OD595 went down just went down to a third within an hour. The OD595 of the cells for control remained stable. However, we could not see the difference between the IPTG induced cells and non-induced cells. This data could suggest that the expression of Antigen43 is not repressed well enough, and is leaking even without being induced. It might also indicate that aggregation can even happen with a small expression.</p>
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<p class="style59">&nbsp;</p>
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<p class="style55"><span class="style53">4-3. Lysis</span></p>
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<p class="style59"><img src="Resources/phototaxisfig11.jpeg" border=0 width=401 height=230 alt="phototaxisfig11" style="vertical-align:baseline"></p>
+
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<p class="style59">This is the result of comparison of lysis gene expression with and without antiholin (fig.8). The yellow line represents control, vector without lysis gene. The blue line shows lysis gene with antiholin and the red line shows lysis gene without antiholin. As you can see, both blue and red line show lower OD660 than the control, indicating that there might be a leakage of lysis gene expression. However system with antiholin kept a higher OD660 than the system without antiholin. Therefore, we concluded that the inducible lysis system consisting of T4 endolysin and holin, functions better with the presence of antiholn.</p>
+
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<p class="style59"><img src="Resources/phototaxisfig12.jpeg" border=0 width=415 height=211 alt="phototaxisfig12" style="vertical-align:baseline"></p>
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<p class="style59">This is the result of lysis gene expression with antiholin, induced by IPTG at stationary phase. Both of these cultivation curves indicate E. coli with lysis genes with antiholin. IPTG was not added to the yellow culture, and IPTG (f.c. 3mM) was added to the blue culture. Within 2 hours after addition of IPTG, the OD660 went down by 80%, indicating that the lysis genes were successfully induced by IPTG. </p>
+
<p class="style59">&nbsp;</p>
<p class="style59">&nbsp;</p>
 +
<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>
 +
<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>
 +
<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>
 +
<p class="style6"><img src="Resources/metallothionein6a.jpeg" border=0 width=566 height=311 alt="metallothionein6" style="vertical-align:baseline"></p>
 +
<p class="style6">&nbsp;</p>
 +
<p class="style6">&nbsp;</p>
 +
<p class="style56">3. Result</p>
 +
<p class="style6"><img src="Resources/metallothioneinr.jpeg" border=0 width=613 height=792 alt="metallothioneinresults" style="vertical-align:baseline"></p>
 +
<p class="style49"><img src="Resources/metallothionein8a.jpeg" border=0 width=495 height=316 alt="metallothionein8" style="vertical-align:baseline"></p>
 +
<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 mM , 300 mM and 400 mM Cd(II) concentration, there is no difference in the OD595 between </span><em><span class="style18">E. coli.</span></em><span class="style18"> However, at 240 mM 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 mM 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 mM, 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</span>.</p>
 +
<p class="style52">&nbsp;</p>
 +
<p class="style52">&nbsp;</p>
 +
<p class="style48"><span class="style57">4. Summary</span></p>
 +
<p class="style40">TBD-                                    As Cd(II)  concentration goes up, growth of E. coli starts to slow down. At 270 mM , 300 mM and 400 mM Cd(II) concentration, there is no difference in the OD595 between E. coli. However, at 240 mM 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 mM 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 mM, 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.</p>
<p class="style59">&nbsp;</p>
<p class="style59">&nbsp;</p>
-
<p class="style61">5. Summary</p>
+
<p class="style49">&nbsp;</p>
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<p class="style55">&nbsp;</p>
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<p class="style56">5. Reference</p>
-
<p class="style55">&nbsp;</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="style55">&nbsp;</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>
-
<p class="style61">6. Reference</p>
+
-
<p class="style59">[1] Jung et al. (2001) An Archeaeal photosignal-Transducing Module Mediates Phototaxis in <em>Escherichia coli. J. Bacteriol.,</em> 183, 6365-6371.</p>
+
-
<p class="style59">[2] Vishwa et al. (2003) Photostimulation of a Sensory Rhodopsin ll/Htrll/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro. <em>Biochemistry,</em> 42, 13887-13892.</p>
+
-
<p class="style59">[3] Kjærgaard et al. (2000) Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens. <em>J. Bacteriol</em>., 182 (17) 4789&#8211;4796.</p>
+
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<p class="style59">[4] Young et al. (2000) Phages will out: strategies of host cell lysis. <em>Trends Microbiol,</em> 8 (3) 120-8.</p>
+
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<p class="style59">[5] Tran et al. (2005) Periplasmic Domains Define Holin-Antiholin Interactions in T4 Lysis Inhibition. <em>J. Bacteriol.</em>, 187 (19) 6631-6640.</p>
+
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<p class="style59 f-lp">[6] Engebrechi and Silverman (1984) Identification of genes and gene products necessary for bacterial bioluminescence. <em>Proc. Natl. Acad. Sci. USA</em>., 81 (13) 4154-4158.</p>
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Revision as of 02:13, 5 October 2011

<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN" "http://www.w3.org/TR/html4/loose.dtd"> Metallothionein v2

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

metallothioneinseq2

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

Fw primer: AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC

Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG

 

metallothioneinseq1

2-2. Cloning MntH

MntH sequence shown in green

Restriction sites shown in pink

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

metallothionein8

As Cd(II) concentration goes up, growth of E. coli starts to slow down. At 270 mM , 300 mM and 400 mM Cd(II) concentration, there is no difference in the OD595 between E. coli. However, at 240 mM 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 mM 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 mM, 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.

 

 

4. Summary

TBD- As Cd(II) concentration goes up, growth of E. coli starts to slow down. At 270 mM , 300 mM and 400 mM Cd(II) concentration, there is no difference in the OD595 between E. coli. However, at 240 mM 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 mM 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 mM, 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.

 

 

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