Team:Tokyo-NoKoGen/metallothionein
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- | <td height= | + | <td height=3356 rowspan=2><p class="style30">Metallothionein - metal-binding peptide</p> |
<p class="style6"><strong>Background</strong></p> | <p class="style6"><strong>Background</strong></p> | ||
<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> | <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> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/7/74/Metallothionein1.jpg" border=0 width=451 height=225 alt="metallothionein1" style="vertical-align:baseline"></p> |
<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 – 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> | <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 – 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> | ||
<p class="style26"> </p> | <p class="style26"> </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> | <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> | ||
<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> | <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> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/d/dd/Metallothionein2.jpg" border=0 width=553 height=220 alt="metallothionein2" style="vertical-align:baseline"></p> |
<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> | <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> | ||
<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> | <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> | ||
<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> | <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> | ||
- | <p class="style37"><img src=" | + | <p class="style37"><img src="https://static.igem.org/mediawiki/2011/6/60/Metallothionein3.jpg" border=0 width=484 height=268 alt="metallothionein3" style="vertical-align:baseline"></p> |
<p class="style26"> </p> | <p class="style26"> </p> | ||
<p class="style26">Method</p> | <p class="style26">Method</p> | ||
<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> | <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> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/1/10/Metallothionein4.jpg" border=0 width=491 height=221 alt="metallothionein4" style="vertical-align:baseline"></p> |
<p class="style6">1.) Design primers to clone SmtA and MntH by PCR</p> | <p class="style6">1.) Design primers to clone SmtA and MntH by PCR</p> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/8/84/Metallothionein5.jpg" border=0 width=407 height=96 alt="metallothionein5" style="vertical-align:baseline"></p> |
<p class="style6"> </p> | <p class="style6"> </p> | ||
<p class="style6"><strong>Cloning SmtA from Synechococcus sp. PCC7942</strong></p> | <p class="style6"><strong>Cloning SmtA from Synechococcus sp. PCC7942</strong></p> | ||
<p class="style6">SmtA sequence shown in red</p> | <p class="style6">SmtA sequence shown in red</p> | ||
<p class="style6">Restriction sites shown in pink</p> | <p class="style6">Restriction sites shown in pink</p> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/9/9c/Metallothionein6.jpg" border=0 width=492 height=222 alt="metallothionein6" style="vertical-align:baseline"></p> |
<p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI. </p> | <p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI. </p> | ||
<p class="style6">Fw primer: AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC</p> | <p class="style6">Fw primer: AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC</p> | ||
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<p class="style6">MntH sequence shown in green</p> | <p class="style6">MntH sequence shown in green</p> | ||
<p class="style6">Restriction sites shown in pink</p> | <p class="style6">Restriction sites shown in pink</p> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/5/58/Metallothionein7.jpg" border=0 width=362 height=180 alt="metallothionein7" style="vertical-align:baseline"></p> |
<p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI.</p> | <p class="style6">Primers to clone and add restriction sites EcoRI, XbaI and SpeI.</p> | ||
<p class="style6">Fw primer: AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC</p> | <p class="style6">Fw primer: AGAATTCGCGGCCGCATCTAGAGAATTTTTTTGC</p> | ||
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<p class="style6">3.) Characterize the effect of expressing SmtA and MntH in E.coli cultured in Cd(II) and As(III) containing medium.</p> | <p class="style6">3.) Characterize the effect of expressing SmtA and MntH in E.coli cultured in Cd(II) and As(III) containing medium.</p> | ||
<p class="style6">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. </p> | <p class="style6">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. </p> | ||
- | <p class="style6"> <img src=" | + | <p class="style6"> <img src="https://static.igem.org/mediawiki/2011/c/c8/Metallothionein8.jpg" border=0 width=494 height=247 alt="metallothionein8" style="vertical-align:baseline"></p> |
<p class="style6"> </p> | <p class="style6"> </p> | ||
<p class="style26">Result</p> | <p class="style26">Result</p> | ||
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Revision as of 11:58, 27 September 2011
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Tokyo-NokoGen 2011 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology |
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Metallothionein - metal-binding peptide 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.
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.
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.
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.
1.) Design primers to clone SmtA and MntH by PCR
Cloning SmtA from Synechococcus sp. PCC7942 SmtA sequence shown in red Restriction sites shown in pink
Primers to clone and add restriction sites EcoRI, XbaI and SpeI. Fw primer: AGAATTCGCGGCCGCATCTAGATGACCTCAACAACGTTGGTC Rv primer: GCTACTAGTATTAGCCGTGGCAGTTACAG
Cloning MntH MntH sequence shown in green Restriction sites shown in pink
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.) 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. 3.) Characterize the effect of expressing SmtA and MntH in E.coli cultured in Cd(II) and As(III) containing medium. 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.
Result
Summary
Reference [1] “Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene”, Koji sode et al., J Mar Biotechnol (1998) [2] “Identification of Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter” Hortence Makui et al., Molecular Microbiology (2000) |
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