Difference between revisions of "Team:NTNU Trondheim/Parts"

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                                    Protocols
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                                    Human practice
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                                    Attributions
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                                    Sponsors
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                                </span>
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                        </li>
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                                    Achievements
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                            <a href='https://2017.igem.org/Team:NTNU_Trondheim/Applied_Design'>
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                                <span>
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                                    Applied Design
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                                </span>
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                            </a>
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                        <li>
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                                <span>
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                                    Hardware
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                                </span>
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                    </ul>
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            <!-- example of slideshow of 1, ie. only a picture. If you wish to make slideshow again, remember to comment
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            back the slideshow.js script in the head section-->
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        </div>
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    </div>
  
  
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    <div class="content">
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        <div class="break_header">
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        </div>
  
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<div class="column full_size">
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        <div class="paragraph_no_img">
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            <h1>Parts</h1>
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            <p>
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                Overview of parts in this project:
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            </p>
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            <table border="1">
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                <tr>
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                    <td><b>Medal</b></td>
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                    <td><b>Part name</b></td>
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                    <td><b>Type</b></td>
 +
                    <td><b>Description</b></td>
 +
                    <td><b>Length (bp)</b></td>
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                </tr>
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                <tr>
 +
                    <td>Bronze</td>
 +
                    <td>BBa_K325909</td>
 +
                    <td>Generator</td>
 +
                    <td>Lux Operon (under pBAD)</td>
 +
                    <td>7637</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Silver</td>
 +
                    <td>BBa_K2265000</td>
 +
                    <td>Device</td>
 +
                    <td>MP6 mutator (original)</td>
 +
                    <td>4908</td>
 +
                </tr>
 +
            </table>
 +
            <p><br><br>
 +
                Our team improved the characterization of the BioBrick <a href="http://parts.igem.org/Part:BBa_K325909">BBa_K325909</a>
 +
                and designed and characterized the BioBrick <a href="http://parts.igem.org/Part:BBa_K2265000">BBa_K2265000</a>.
 +
                Both are documented in the iGEM Registry:
 +
                <br><br>
 +
                <a href="http://parts.igem.org/Part:BBa_K325909">BBa_K325909</a>
 +
                <br><br>
 +
                <a href="http://parts.igem.org/Part:BBa_K2265000">BBa_K2265000</a>
 +
            </p>
 +
        </div>
  
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<h1>Parts</h1>
 
  
−
<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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        <div class="break_2">
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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            <img src="https://static.igem.org/mediawiki/2017/5/52/T--NTNU_Trondheim--splitter_2.svg">
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        </div>
  
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        <div class="paragraph_no_img">
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            <h1>Biobrick contribution: The MP6 mutator (original)</h1>
 +
            <p>
 +
                The MP6 mutator (original) biobrick is a plasmid with an operon under control of the promoter pBAD
 +
                together with the transcription factor gene AraC, and six other genes that disrupt DNA replication:
 +
                DnaQ926, Dam, SeqA, EmrR, Ugi and Cda1. We designed this biobrick to accelerate bacterial mutation rate,
 +
                which could be useful for our evolution experiments.
 +
                <br><br>
 +
                The DnaQ926 is a dominant negative variant of the <i>Escherichia coli</i> DNA pol III proofreading domain. Dam has a
 +
                strong mutator effect due to impaired mismatch repair. The SeqA protein negatively regulates the initiation
 +
                of DNA replication at the origin of replication. A low expression of SeqA reduces the transcription of
 +
                the MP6 plasmid in absence of arabinose. The EmrR gene codes for a protein that compromises intracellular
 +
                dNTP pools. Ugi codes for a protein that inhibit Ung, a mutagenesis preventing enzyme, through mimicry
 +
                of structural and electronic features of uracil-containing DNA. CDA1 codes for a cytidine deaminase
 +
                that is reported to promote the mutation of both prokaryotic and eukaryotic genomic DNA.[1]
 +
                <br><br>
 +
                The biobrick is based on the MP6 plasmid from addgene (Addgene plasmid # 69669). The original plasmid
 +
                was changed in order to make it a biobrick. The change done to the plasmid was removing a Spel restriction
 +
                site by changing an A to a C (nucleotide number 4971 in the original MP6 plasmid). A biobrick prefix was
 +
                also added in front of the araC gene and a biobrick suffix after the CDA1 gene.
 +
                <br><br><br>                 
 +
                <u>Source:</u><br>
 +
                [1] Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Badran AH, Liu DR. 
 +
                Nat Commun. 2015 Oct 7;6:8425. doi: 10.1038/ncomms9425. 10.1038/ncomms9425 PubMed 26443021
 +
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            <h1>Characterization of the Lux Operon biobrick</h1>
 +
            <p>
 +
                We chose to improve this biobrick as we originally wanted to use the lux operon as a reporter for our
 +
                arabinose-inducible system. In this way, we could test how the expression from the pBAD promoter was
 +
                dependent on arabinose concentration.
 +
                <br><br>
 +
                By looking at previous characterisation results of the biobrick, we thought it would work reasonably to
 +
                use DH5α competent <i>E. coli</i> cells and an incubation temperature of 37 °C. However, when testing with
 +
                0.01M arabinose concentrations, no observable luminescence was detected at any time after induction.
 +
                Therefore, we decided to characterise the biobrick by measuring the luminescence from the lux operon
 +
                at different arabinose concentrations and temperatures. We measured the luminescence with a Tecan M200
 +
                plate reader, using four different temperatures and six different arabinose concentrations. As the
 +
                machine could only perform heating and not cooling, it was challenging to measure at temperatures
 +
                below 29 °C.
 +
                <br><br>
 +
                The results are given on the biobrick <a href="http://parts.igem.org/Part:BBa_K2265000">parts page</a>.
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            </p>
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        </div>
  
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<a href="http://www.ntnu.edu/ibt" target="_blank">
 +
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                    </a>
 +
                    <a href="http://www.biokjemisk.no/" target="_blank">
 +
                        <img src="https://static.igem.org/mediawiki/2017/f/f4/T--NTNU_Trondheim--NBS_forslag1_transparent.png">
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 +
<a href="http://www.vectronbiosolutions.com/" target="_blank">
 +
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 +
<a href="http://digitallifenorway.org/gb/" target="_blank">
 +
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 +
                    <a href="mailto:igem-team@ntnu.no" >
 +
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                    <p>
 +
                        Email: igem-team@ntnu.no
 +
                    </p>
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                </div>
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            </div>
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<h5>Note</h5>
 
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
 
 
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<p>The information needed to initially create a part on the Registry is:</p>
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<li>Part Name</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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Revision as of 00:02, 2 November 2017

Parts

Overview of parts in this project:

Medal Part name Type Description Length (bp)
Bronze BBa_K325909 Generator Lux Operon (under pBAD) 7637
Silver BBa_K2265000 Device MP6 mutator (original) 4908



Our team improved the characterization of the BioBrick BBa_K325909 and designed and characterized the BioBrick BBa_K2265000. Both are documented in the iGEM Registry:

BBa_K325909

BBa_K2265000

Biobrick contribution: The MP6 mutator (original)

The MP6 mutator (original) biobrick is a plasmid with an operon under control of the promoter pBAD together with the transcription factor gene AraC, and six other genes that disrupt DNA replication: DnaQ926, Dam, SeqA, EmrR, Ugi and Cda1. We designed this biobrick to accelerate bacterial mutation rate, which could be useful for our evolution experiments.

The DnaQ926 is a dominant negative variant of the Escherichia coli DNA pol III proofreading domain. Dam has a strong mutator effect due to impaired mismatch repair. The SeqA protein negatively regulates the initiation of DNA replication at the origin of replication. A low expression of SeqA reduces the transcription of the MP6 plasmid in absence of arabinose. The EmrR gene codes for a protein that compromises intracellular dNTP pools. Ugi codes for a protein that inhibit Ung, a mutagenesis preventing enzyme, through mimicry of structural and electronic features of uracil-containing DNA. CDA1 codes for a cytidine deaminase that is reported to promote the mutation of both prokaryotic and eukaryotic genomic DNA.[1]

The biobrick is based on the MP6 plasmid from addgene (Addgene plasmid # 69669). The original plasmid was changed in order to make it a biobrick. The change done to the plasmid was removing a Spel restriction site by changing an A to a C (nucleotide number 4971 in the original MP6 plasmid). A biobrick prefix was also added in front of the araC gene and a biobrick suffix after the CDA1 gene.


  Source:
[1] Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Badran AH, Liu DR.  Nat Commun. 2015 Oct 7;6:8425. doi: 10.1038/ncomms9425. 10.1038/ncomms9425 PubMed 26443021

Characterization of the Lux Operon biobrick

We chose to improve this biobrick as we originally wanted to use the lux operon as a reporter for our arabinose-inducible system. In this way, we could test how the expression from the pBAD promoter was dependent on arabinose concentration.

By looking at previous characterisation results of the biobrick, we thought it would work reasonably to use DH5α competent E. coli cells and an incubation temperature of 37 °C. However, when testing with 0.01M arabinose concentrations, no observable luminescence was detected at any time after induction. Therefore, we decided to characterise the biobrick by measuring the luminescence from the lux operon at different arabinose concentrations and temperatures. We measured the luminescence with a Tecan M200 plate reader, using four different temperatures and six different arabinose concentrations. As the machine could only perform heating and not cooling, it was challenging to measure at temperatures below 29 °C.

The results are given on the biobrick parts page.

Email: igem-team@ntnu.no