Team:Alberta/Methodology/Parts

From 2011.igem.org

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            METHODOLOGY
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        <h2>Parts</h2>
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        <table class=figure>
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            <tr class=top>
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                <th>Part</th>
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                <th>Registry Number</th>
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                <th>Description</th>
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            </tr>
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            <tr class=odd>
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                <td>FadD 5'-UTR</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612001">BBa_K612001</a></td>
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                <td>This is the 5'-UTR of the FadD gene of <i>N. crassa</i>. It is suitable for creating constructs which can knockout the FadD gene, as well knock in new genes.</td>
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            </tr>
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            <tr>
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                <td>TesA' Whole Gene</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612002">BBa_K612002</a></td>
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                <td>The whole gene construct, with the TesA' open reading frame, the Actin promoter and the TrpC terminator</td>
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            </tr>
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            <tr class=odd>
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                <td>Hygromycin B</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612003">BBa_K612003</a></td>
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                <td>Acts as a selection marker for transformed N.Crass. Gives resistance to the antibiotic Hygromycin</td>
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            </tr>
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            <tr>
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                <td>FadD 3'-UTR</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612004">BBa_K612004</a></td>
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                <td>This is the 3'-UTR of the FadD gene of <i>N. crassa</i>. It is suitable for creating constructs which can knockout the FadD gene, as well knock in new genes.</td>
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            </tr>
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            <tr class=odd>
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                <td>Actin Promoter </td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612005">BBa_K612005</a></td>
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                <td>A constitutive promoter used in the literature for genes transformed into <i>N. crassa</i>. </td>
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            </tr>
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            <tr>
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                <td>TrpC Terminator</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612007">BBa_K612007</a></td>
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                <td>A terminator used for <i>N. crassa</i> genes. It is also used in other fungus species. </td>
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            </tr>
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            <tr class=odd>
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                <td>TesA' CDS</td>
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                <td><a href="http://partsregistry.org/Part:BBa_K612008">BBa_K612008</a></td>
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                <td>The open reading frame for the TesA' gene. This is a thioesterase from <i>E. coli</i> which has been codon optimized for use in <i>N. crassa</i>. This thioesterase preferably cleaves off sixteen carbon fatty acids from the fatty acid synthase complex, producing free fatty acids.  </td>
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            </tr>
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        <br>
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        <h3>Favourite Parts</h3>
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        <p>So far only two parts (the 5'FadD region and the TrpC
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        terminator) have been put in IGEM plasmids for judging. The
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        5' FadD-UTR was made by using the <i>N. crassa</i> genome as a
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        template in a PCR, BsaI cut sites were added to each end
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        using the PCR primers. The TrpC terminator was synthesized
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        with the codon optimized TesA' and was taken out of that
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        part using PCR. The parts plasmids were checked by using
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        NotI and BsaI digestions and running the DNA on a gel. The
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        gels are shown below:</p>
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        <br>
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        <div style="float:left; margin-left: 75px;">
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            <center>
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            <h4>5' FadD-UTR</h4>
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            </center>
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        <img src=https://static.igem.org/mediawiki/2011/0/01/Parts-gel-fadD.png>
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        </div>
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            <center>
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            <h4>TrpC Terminator</h4>
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            </center>
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        <img width=250px src=https://static.igem.org/mediawiki/2011/8/8c/Parts-gel-trpC.png>
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Latest revision as of 21:37, 28 September 2011

METHODOLOGY

Parts

Part Registry Number Description
FadD 5'-UTR BBa_K612001 This is the 5'-UTR of the FadD gene of N. crassa. It is suitable for creating constructs which can knockout the FadD gene, as well knock in new genes.
TesA' Whole Gene BBa_K612002 The whole gene construct, with the TesA' open reading frame, the Actin promoter and the TrpC terminator
Hygromycin B BBa_K612003 Acts as a selection marker for transformed N.Crass. Gives resistance to the antibiotic Hygromycin
FadD 3'-UTR BBa_K612004 This is the 3'-UTR of the FadD gene of N. crassa. It is suitable for creating constructs which can knockout the FadD gene, as well knock in new genes.
Actin Promoter BBa_K612005 A constitutive promoter used in the literature for genes transformed into N. crassa.
TrpC Terminator BBa_K612007 A terminator used for N. crassa genes. It is also used in other fungus species.
TesA' CDS BBa_K612008 The open reading frame for the TesA' gene. This is a thioesterase from E. coli which has been codon optimized for use in N. crassa. This thioesterase preferably cleaves off sixteen carbon fatty acids from the fatty acid synthase complex, producing free fatty acids.

Favourite Parts

So far only two parts (the 5'FadD region and the TrpC terminator) have been put in IGEM plasmids for judging. The 5' FadD-UTR was made by using the N. crassa genome as a template in a PCR, BsaI cut sites were added to each end using the PCR primers. The TrpC terminator was synthesized with the codon optimized TesA' and was taken out of that part using PCR. The parts plasmids were checked by using NotI and BsaI digestions and running the DNA on a gel. The gels are shown below:


5' FadD-UTR

TrpC Terminator

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