Difference between revisions of "Team:NCKU Tainan/Parts"

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                            <a class="nav-link" href="#Subtitle1">Overview</a>
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            <a class="nav-link" href="#Subtitle1">Parts</a>
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                            <a class="nav-link" href="#Subtitle2">Experimental Approach & Results</a>
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            <a class="nav-link" href="#Subtitle2">Composite Parts</a>
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                            <a class="nav-link" href="#Subtitle1">Reference</a>
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          <h1>Parts</h1>
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                    <h1>Improve</h1>
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          <div class="divider"></div>
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                    <h2 id ="Subtitle1">Overview</h2>
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                        <p>    In our project, one of the major goals is to reduce the uremic toxin, p-Cresol. Clostridium bacteria inside the gut will ferment excess tyrosine into p-Cresol. With the help of <a href="https://2019.igem.org/Team:NCKU_Tainan/Parts">Tyrosine ammonia-lyase (TAL)</a> , we are able to convert this excess tyrosine into a harmless product, p-Coumaric acid instead of the toxic p-Cresol.  As such, the concentration of p-Cresol (mainly produced by C. difficile) can be reduced.</p>
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                        <p>    For our construct design, we focused on <a href="http://parts.igem.org/Part:BBa_I742148">BioBrick BBa_I742148</a>from the <a href="https://2007.igem.org/Edinburgh">2007 iGEM Edinburgh team</a><sup>[<a href="#ref1">1</a>]</sup> in the iGEM BioBrick library. This enzyme has been shown to have the highest Km value compared to other TAL enzyme. </p>
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                        <p>    We hypothesized that the native ribosome binding site(NRBS) from Saccharothrix espanaensis will not have a high translation efficiency in E.coli Nissle, so we changed the NRBS in front of TAL into a strong ribosome binding site B0034. Also, we noticed that there was no spacer sequence between NRBS and the TAL coding sequence(CDS) in <a href="http://parts.igem.org/Part:BBa_I742148">BBa_I742148</a>. </p>
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                        <p>    According to research, the length of the spacer sequence between RBS and CDS strongly affects translation efficiency.<sup>[<a href="#ref2">2</a>]</sup>  We decided to follow the standard iGEM BioBrick assembly rule to add 6bp of scar sequence between RBS and CDS. The schematic below depicts how we improved the BioBrick.</p>
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                </section>
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                <hr>
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                <section>
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            <!--section name:Experimental Approach & Results-->
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                    <h2 id ="Subtitle2">Experimental Approach & Results</h2>
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                        <p>    To prove that TAL expression was improved with the change of RBS (<a href="http://parts.igem.org/Part:BBa_I742146">I742146</a> into B0034), we checked the protein expression level by SDS-PAGE and measured the amount of p-coumaric acid production normalized by O.D. value. </p>
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                        <p>    The result of SDS-PAGE is shown below whereby the expected protein size for TAL is 54kDa.</p>
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                        <p>    As seen in the results above, there’s no distinguishable band around the expected size. Since we cannot identify any significant protein bands on SDS gel, we performed an RT-PCR experiment to confirm that the constructed BioBrick is being transcribed. The result is shown below.</p>
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                        <p>    As shown in Fig.3(b), the cDNA for both TAL constructs are being detected by RT-PCR. Hence, it confirms that the TAL genes are actually being transcribed in E.coli Nissle.</p>
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                        <p>    Finally, to determine the protein activity of TAL, we performed a functional test using the n-octanol extraction method (hyperlink: TAL and TyrP functional test), which was previously proposed by the <a href="https://2013.igem.org/Team:Uppsala">2013 iGEM Uppsala team</a> <sup>[<a href="#ref3">3</a>]</sup>and has been verified by HPLC. The p-Coumaric acid concentration was measured through the absorbance value at 310nm wavelength under Nanodrop UV-Vis wavelength. </p>
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                        <p>    We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa_K2997009 and BBa_K2997010) to determine if p-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig 6., BBa_K2997010 is able to produce a higher amount of p-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into p-Coumaric acid can increase by 1.73-fold.  Therefore, we have shown that we have improved a previous BioBrick.</p>
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                        <p>    For more information, please visit our <a href="https://2019.igem.org/Team:NCKU_Tainan/Results">Results page.</a></p>
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                </section>
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                <section class="ref">
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                    <h2>References</h2>
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                    <ol>
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                        <li id="ref1">2007 iGEM Edinburgh Team </li>
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                        <li id="ref2">Chen, H., Bjerknes, M., Kumar, R., & Jay, E. (1994, November 25). Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523762/. </li>
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                        <li id="ref3">2013 iGEM Uppsala Team</li>
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                    </ol>
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                </section>
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            </main>
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         </div>
 
         </div>
 +
        <!--overview part-->
 +
        <div style="margin-top:7%;">
 +
          <h2>Overview</h2>
 +
          <p style="padding:1rem;padding-right: 10%;text-align: justify;">&emsp;&emsp;BioBricks have always been the foundational building blocks for iGEM projects. When constructing our BioBricks, we made use of the IDT synthesis offer to generate our required parts in a fast and efficient way. Additionally, we made use of PCR amplification with our constructed expression plasmids as template to construct several BioBricks. All of the parts contain a coding sequence, some of which extended with promoters or introns. We created a part collection comprised of 15 basic parts and 6 improved composite part. All parts were generated during our project.
 +
          </p>
 +
        </div>
 +
        <!--parts-->
 +
        <section>
 +
          <h2 id ="Subtitle1">Parts table</h2>
 +
          <!--If you want to divide it into two block use below part-->
 +
          <div class="table-responsive" style="overflow-x:auto;">
 +
            <table class="table table-bordered table-hover">
 +
              <thead>
 +
                <tr>
 +
                  <th scope="col">Name</th>
 +
                  <th scope="col">Description</th>
 +
                  <th scope="col">Type</th>
 +
                  <th scope="col">Length</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a></th>
 +
                  <td>Constitutive promoter (P<sub>J23100</sub>)</td>
 +
                  <td>Promotor</td>
 +
                  <td>35bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K1123000">BBa_K1123000</a></th>
 +
                  <td>FNR promoter (P<sub>fnr</sub>)</td>
 +
                  <td>Promoter</td>
 +
                  <td>120bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a></th>
 +
                  <td>Strong promoter J23100 and strong RBS B0034</td>
 +
                  <td>Promoter+RBS</td>
 +
                  <td>55bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_I742146">BBa_ I742146</a></th>
 +
                  <td>Tyrosine ammonia-lyase native RBS (N<sub>RBS</sub>)</td>
 +
                  <td>RBS</td>
 +
                  <td>14bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_B0017">BBa_B0017</a></th>
 +
                  <td>Double terminator (B0010-B0010)</td>
 +
                  <td>Terminator</td>
 +
                  <td>160bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997000">BBa_K2997000</a></th>
 +
                  <td>Tyrosine transporter(tyrP)</td>
 +
                  <td>Coding</td>
 +
                  <td>1297bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_I742142">BBa_I1742142</a></th>
 +
                  <td>Tyrosine ammonia-lyase (sam8)</td>
 +
                  <td>Coding</td>
 +
                  <td>1536bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997001">BBa_K2997001</a></th>
 +
                  <td>Bacteriocin Full length(CBM-B)</td>
 +
                  <td>Coding</td>
 +
                  <td>867bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997002">BBa_K2997002</a></th>
 +
                  <td>Bacteriocin short length(sCBM-B)</td>
 +
                  <td>Coding</td>
 +
                  <td>252bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997003">BBa_K2997003</a></th>
 +
                  <td>Sigma 54-dependent transcriptional regulator (pchR)</td>
 +
                  <td>Coding</td>
 +
                  <td>1854bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997004">BBa_K2997004</a></th>
 +
                  <td>PchR binding range</td>
 +
                  <td>Coding</td>
 +
                  <td>567bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_J18921">BBa_J18921</a></th>
 +
                  <td>6aa [GS]x linker</td>
 +
                  <td>Coding</td>
 +
                  <td>18bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K1610300">BBa_K1610300</a></th>
 +
                  <td>Secretion tag (YebF)</td>
 +
                  <td>Coding</td>
 +
                  <td>354bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a></th>
 +
                  <td>Wild type green fluorescence protein (wtgfp)</td>
 +
                  <td>Coding</td>
 +
                  <td>720bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2762017">BBa_K2762017</a></th>
 +
                  <td>Superfolder green fluorescence protein (sfgfp)</td>
 +
                  <td>Coding</td>
 +
                  <td>720bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </div>
 +
        </section>
 +
        <!--comsposite parts-->
 +
        <section style="margin-bottom:5%;">
 +
          <h2 id ="Subtitle2">Composite Parts table</h2>
 +
          <div class="table-responsive" style="overflow-x:auto;">
 +
            <table class="table table-bordered">
 +
              <thead>
 +
                <tr>
 +
                  <th scope="col">Name</th>
 +
                  <th scope="col">Description</th>
 +
                  <th scope="col">Type</th>
 +
                  <th scope="col">Length</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997005">BBa_K2997005</a></th>
 +
                  <td>K880005-yebF-GS-sCBMB</td>
 +
                  <td style="vertical-align:middle" rowspan="8">Composite</td>
 +
                  <td>685bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997006">BBa_K2997006</a></th>
 +
                  <td>K880005-yebF-TB-sCBMB</td>
 +
                  <td>120bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997007">BBa_K2997007</a></th>
 +
                  <td>K880005-yebF-CBMB</td>
 +
                  <td>1282bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997008">BBa_K2997008</a></th>
 +
                  <td>Native promoter-PchR-sfgfp</td>
 +
                  <td>3137bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997009">BBa_K2997009</a></th>
 +
                  <td>J23100-NRBS-sam8</td>
 +
                  <td>1606bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997010">BBa_K2997010</a></th>
 +
                  <td>K880005-sam8</td>
 +
                  <td>1613bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_K2997011">BBa_K2997011</a></th>
 +
                  <td>B0034-sam8</td>
 +
                  <td>1572bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <th scope="row"><a target="blank" href="http://parts.igem.org/Part:BBa_I742106">BBa_I742106</a></th>
 +
                  <td>Sam8 with ribosome binding site</td>
 +
                  <td>1550bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </div>
 +
        </section>
 +
      </main>
 
     </div>
 
     </div>
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  </div>
 
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Revision as of 14:51, 19 October 2019

>

Parts

Overview

  BioBricks have always been the foundational building blocks for iGEM projects. When constructing our BioBricks, we made use of the IDT synthesis offer to generate our required parts in a fast and efficient way. Additionally, we made use of PCR amplification with our constructed expression plasmids as template to construct several BioBricks. All of the parts contain a coding sequence, some of which extended with promoters or introns. We created a part collection comprised of 15 basic parts and 6 improved composite part. All parts were generated during our project.

Parts table

Name Description Type Length
BBa_J23100 Constitutive promoter (PJ23100) Promotor 35bp
BBa_K1123000 FNR promoter (Pfnr) Promoter 120bp
BBa_K880005 Strong promoter J23100 and strong RBS B0034 Promoter+RBS 55bp
BBa_ I742146 Tyrosine ammonia-lyase native RBS (NRBS) RBS 14bp
BBa_B0017 Double terminator (B0010-B0010) Terminator 160bp
BBa_K2997000 Tyrosine transporter(tyrP) Coding 1297bp
BBa_I1742142 Tyrosine ammonia-lyase (sam8) Coding 1536bp
BBa_K2997001 Bacteriocin Full length(CBM-B) Coding 867bp
BBa_K2997002 Bacteriocin short length(sCBM-B) Coding 252bp
BBa_K2997003 Sigma 54-dependent transcriptional regulator (pchR) Coding 1854bp
BBa_K2997004 PchR binding range Coding 567bp
BBa_J18921 6aa [GS]x linker Coding 18bp
BBa_K1610300 Secretion tag (YebF) Coding 354bp
BBa_E0040 Wild type green fluorescence protein (wtgfp) Coding 720bp
BBa_K2762017 Superfolder green fluorescence protein (sfgfp) Coding 720bp

Composite Parts table

Name Description Type Length
BBa_K2997005 K880005-yebF-GS-sCBMB Composite 685bp
BBa_K2997006 K880005-yebF-TB-sCBMB 120bp
BBa_K2997007 K880005-yebF-CBMB 1282bp
BBa_K2997008 Native promoter-PchR-sfgfp 3137bp
BBa_K2997009 J23100-NRBS-sam8 1606bp
BBa_K2997010 K880005-sam8 1613bp
BBa_K2997011 B0034-sam8 1572bp
BBa_I742106 Sam8 with ribosome binding site 1550bp

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