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

 
(26 intermediate revisions by 4 users not shown)
Line 22: Line 22:
 
                         </li>
 
                         </li>
 
                         <li class="nav-item">
 
                         <li class="nav-item">
−
                             <a class="nav-link" href="#Subtitle2">Experimental Results</a>
+
                             <a class="nav-link" href="#Subtitle2">Results</a>
 
                         </li>
 
                         </li>
 
                     </ul>
 
                     </ul>
Line 36: Line 36:
 
             <!--section name:Overview-->
 
             <!--section name:Overview-->
 
                     <h2 id ="Subtitle1">Overview</h2>
 
                     <h2 id ="Subtitle1">Overview</h2>
−
                         <p>   In our project, one of the major goals is to reduce the uremic toxin, <i>p</i>-Cresol. <i>Clostridium</i> bacteria inside the gut will ferment excess tyrosine into <i>p</i>-Cresol. With the help of <a href="https://2019.igem.org/Team:NCKU_Tainan/Parts" target="_blank">tyrosine ammonia-lyase (TAL)</a> , we are able to convert this excess tyrosine into a harmless product, <i>p</i>-Coumaric acid instead of the toxic <i>p</i>-Cresol.  As such, the concentration of <i>p</i>-Cresol can be reduced.</p>
+
                         <p>&emsp;&emsp;In our project, the superfolder green fluorescent protein (sfGFP) allows better quantification of promoter strength and sensitivity<sup>[<a href="#ref1">1</a>,<a href="#ref2">2</a>]</sup>. In the oxidative stress sensing system biobrick, we improved the <a href="http://parts.igem.org/Part:BBa_K2610031">biobrick BBa_K2610031</a> from the <a href="https://2019.igem.org/Team:Leiden">2019 iGEM Leiden team</a> by changing GFP (<a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a>)<sup>[<a href="#ref2">2</a>]</sup> into sfGFP(<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), which is hypothesized to have a higher expression level than GFP [Fig. 1]. We also added transcription activator SoxR to our biobrick for increased function of the oxidative stress sensing system<sup>[<a href="#ref3">3</a>]</sup>.
−
                        <p>    For our construct design, we focused on <a href="http://parts.igem.org/Part:BBa_I742148" target="_blank">BioBrick BBa_I742148</a> from the <a href="https://2007.igem.org/Edinburgh" target="_blank">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>
+
                  <div class="container-fluid p-0">
−
                        <p>    We hypothesized that the native ribosome binding site (NRBS) from <i>Saccharothrix espanaensis</i> will not have a high translation efficiency in <i>E. coli</i> 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" target="_blank">BBa_I742148</a>. </p>
+
−
                        <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 6 bp of scar sequence between RBS and CDS. The schematic below depicts how we improved the BioBrick.</p>
+
−
                        <div class="container-fluid p-0">
+
 
                             <div class="row no-gutters">
 
                             <div class="row no-gutters">
 
                                 <div class="col-lg ">
 
                                 <div class="col-lg ">
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                         <!-- put link in href & src , width in a can adjust-->
 
                                         <!-- put link in href & src , width in a can adjust-->
−
                                         <a href="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" alt="" title="" style="width:100%"></a>
+
                                         <a href="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" target="_blank" style="width:70%"><img src="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" alt="" title="" style="width:100%"></a>
−
                                         <figcaption>Fig. 1. A schematic depicting our approach.</figcaption>
+
                                         <figcaption>Fig.1. Improvement big picture</figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
Line 55: Line 52:
 
                 <section>
 
                 <section>
 
             <!--section name:Experimental Approach & Results-->
 
             <!--section name:Experimental Approach & Results-->
−
                     <h2 id ="Subtitle2">Experimental Results</h2>
+
                     <h2 id ="Subtitle2">Results</h2>
−
                        <p>   We wanted to prove that TAL expression was improved with the change of RBS (<a href="http://parts.igem.org/Part:BBa_I742146" target="_blank">BBa_I742146</a> into B0034). First, RT-PCR experiment was used to confirm that the constructed TAL Biobrick is being transcribed. As seen in Fig.2, cDNA for both bacteria carrying TAL constructs are being detected by PCR, thus confirming that the TAL genes is actually being transcribed in <i>E. coli</i> Nissle.</p>
+
                  <h3>Disk assay</h3>
−
                        <div class="container-fluid p-0">
+
                        <p>&emsp;&emsp;Disk assay was used to check the effect of each inducer. The concentration of the different inducers we used are listed below:</p>  
 +
                  <div class="container-fluid p-0">
 
                             <div class="row no-gutters">
 
                             <div class="row no-gutters">
 
                                 <div class="col-lg ">
 
                                 <div class="col-lg ">
−
                                    <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
−
                                        <!-- put link in href & src , width in a can adjust-->
 
−
                                        <a href="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" target="_blank" style="width:70%"><img src="https://static.igem.org/mediawiki/2021/a/a0/T--NCKU_Tainan--improvement.png" alt="" title="" style="width:100%"></a>
 
−
                                    </figure>
 
 
                                     <div class="table-responsive" style="overflow-x:auto;">
 
                                     <div class="table-responsive" style="overflow-x:auto;">
−
                                         <table class="table-bordered table-striped" style="word-wrap:break-word;">
+
                                         <table class="table-bordered table-striped" style="word-wrap:break-word; width:50%">
 
                                             <thead>
 
                                             <thead>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <th style="text-align: center">Lane</th>
+
                                                     <th style="text-align: center">Inducer</th>
−
                                                     <th style="text-align: center">Template</th>
+
                                                     <th style="text-align: center">Volume per disk</th>
−
                                                    <th style="text-align: center">Primers</th>
+
−
                                                    <th style="text-align: center">Lane</th>
+
−
                                                    <th style="text-align: center">Template</th>
+
−
                                                    <th style="text-align: center">Primers</th>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                             </thead>
 
                                             </thead>
 
                                             <tbody>
 
                                             <tbody>
 
                                                 <tr>
 
                                                 <tr>
−
                                                    <td style="text-align: center">1</td>
+
                                                  <td style="text-align: center">H<sub>2</sub>O<sub>2</sub> (30%)</td>
−
                                                    <td>PCR positive control</td>
+
                                                 
−
                                                    <td></td>
+
                                                     <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">8</td>
+
                                                 
−
                                                    <td>BBa_K2997009 cDNA</td>
+
−
                                                     <td style="text-align: center">3+4</td>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <td style="text-align: center">2</td>
+
                                                     <td style="text-align: center">H<sub>2</sub>O<sub>2</sub> (3%)</td>
−
                                                    <td>BBa_K2997009 cDNA</td>
+
                                                     <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">1+2</td>
+
−
                                                    <td style="text-align: center">9</td>
+
−
                                                    <td>BBa_K2997010 cDNA</td>
+
−
                                                     <td style="text-align: center">3+4</td>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <td style="text-align: center">3</td>
+
                                                     <td style="text-align: center">MD (menadione)(10 mM)</td>
−
                                                    <td>BBa_K2997009 RNA</td>
+
                                                        <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">1+2</td>
+
−
                                                    <td style="text-align: center">10</td>
+
−
                                                    <td>BBa_K2997009 RNA</td>
+
−
                                                    <td style="text-align: center">3+4</td>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <td style="text-align: center">4</td>
+
                                                     <td style="text-align: center">DMSO (solvent for MD)</td>
−
                                                    <td>BBa_K2997009 plasmid</td>
+
                                                  <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">1+2</td>
+
−
                                                    <td style="text-align: center">11</td>
+
−
                                                    <td>BBa_K2997010 RNA</td>
+
−
                                                    <td style="text-align: center">3+4</td>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <td style="text-align: center">5</td>
+
                                                     <td style="text-align: center">PQ (paraquat)(1 mM)</td>
−
                                                    <td>BBa_K2997010 cDNA</td>
+
                                                      <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">1’+2</td>
+
−
                                                    <td style="text-align: center">12</td>
+
−
                                                    <td>BBa_K2997009 plasmid</td>
+
−
                                                    <td style="text-align: center">3+4</td>
+
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
−
                                                     <td style="text-align: center">6</td>
+
                                                     <td style="text-align: center">MQ (solvent for PQ)</td>
−
                                                    <td>BBa_K2997010 RNA</td>
+
                                                  <td style="text-align: center">10 &mu;l</td>
−
                                                    <td style="text-align: center">1’+2</td>
+
−
                                                    <td style="text-align: center">13</td>
+
−
                                                    <td>BBa_K2997010 plasmid</td>
+
−
                                                    <td style="text-align: center">3+4</td>
+
−
                                                </tr>
+
−
                                                <tr>
+
−
                                                    <td style="text-align: center">7</td>
+
−
                                                    <td>BBa_K2997010 plasmid</td>
+
−
                                                    <td style="text-align: center">1’+2</td>
+
−
                                                    <td></td>
+
−
                                                    <td></td>
+
−
                                                    <td></td>
+
 
                                                 </tr>
 
                                                 </tr>
 +
 
                                             </tbody>
 
                                             </tbody>
 
                                         </table>
 
                                         </table>
 
                                     </div>
 
                                     </div>
−
                                     <figcaption>Fig. 2. Reverse Transcription (RT)-PCR Results to confirm that our construct is being transcribed. (a) Schematics show location of amplified regions and primers. (b) 1.5 % Agarose gel shows PCR results. All products have expected size of 250 bp as shown in (a). Template and primers used in this experiment are listed in the table. (cDNA: Total cDNA; RNA: Total RNA; plasmid: pSB1C3 containing respective Biobrick.)</figcaption>
+
                                     <figcaption>Table 1. Oxidative stress inducers</figcaption>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
  
−
                         <p><p>    Then, we carried out SDS-PAGE to check the protein expression of TAL, both with TyrP and without TyrP. The expected protein size of TAL is 54 kDa and the expected protein size of TyrP is 43 kDa. As seen in the results below, however, there’s no distinguishable band around both sizes.</p>
+
                         <p>&emsp;&emsp;With sfGFP, the induced system result can be checked under UV light, making it easier to tell the difference between each inducer (Fig. 2).</p>
−
                        <div class="container-fluid p-0">
+
 
 +
                  <div class="container-fluid p-0">
 
                             <div class="row no-gutters">
 
                             <div class="row no-gutters">
 
                                 <div class="col-lg ">
 
                                 <div class="col-lg ">
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                         <!-- put link in href & src , width in a can adjust-->
 
                                         <!-- put link in href & src , width in a can adjust-->
−
                                         <a href="https://static.igem.org/mediawiki/2019/0/02/T--NCKU_Tainan--Result-SDSPAGE_2.jpg" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/2019/0/02/T--NCKU_Tainan--Result-SDSPAGE_2.jpg" alt="" title="" style="width:100%"></a>
+
                                         <a href="https://static.igem.org/mediawiki/2021/8/88/T--NCKU_Tainan--PsoxS_Disk_Assay.png" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/2021/8/88/T--NCKU_Tainan--PsoxS_Disk_Assay.png" alt="" title="" style="width:100%"></a>
−
                                         <figcaption>Fig. 3. 12% SDS PAGE of <i>E. coli</i> Nissle 1917 with different plasmids. M: Marker; Lane 1: Wild Type; Lane 2: pSB1C3; Lane 3: BBa_K2997009 ; Lane 4: BBa_K2997010; Lane 5: Dual plasmid containing BBa_K2997009 and BBa_K2997000; Lane 6: Dual plasmid containing BBa_K29970010  and BBa_K2997000 ; Lane 7: Positive control (c.d. 3392)</figcaption>
+
                                         <figcaption>Fig. 2. The induced result is clear to be seen after the improvement.</figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
−
                        <p>   Finally, to confirm the protein activity of TAL and TyrP, we performed a functional test using <a href="https://2019.igem.org/Team:NCKU_Tainan/Design#Subtitle1" target="_blank"><i>n</i>-octanol extraction method</a>, which was previously proposed by iGEM_Uppsala 2013 and has been verified by HPLC<sup>[<a href="#ref3">3</a>]</sup>. The <i>p</i>-Coumaric acid concentration was measured through the absorbance value at 310 nm wavelength under Nanodrop UV-Vis wavelength. </p>
+
                  <h3>Oxidative Stress Assay</h3>
 
                         <div class="container-fluid p-0">
 
                         <div class="container-fluid p-0">
 
                             <div class="row no-gutters">
 
                             <div class="row no-gutters">
Line 159: Line 121:
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                     <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 
                                         <!-- put link in href & src , width in a can adjust-->
 
                                         <!-- put link in href & src , width in a can adjust-->
−
                                         <a href="https://static.igem.org/mediawiki/2019/d/d3/T--NCKU_Tainan--Results_tyrosine_to_pCA%2C_RBS_improve.png" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/2019/d/d3/T--NCKU_Tainan--Results_tyrosine_to_pCA%2C_RBS_improve.png" alt="" title="" style="width:100%"></a>
+
                                         <a href="https://static.igem.org/mediawiki/2021/0/0b/T--NCKU_Tainan--improvement2.png" target="_blank" style="width:70%"><img src="https://static.igem.org/mediawiki/2021/0/0b/T--NCKU_Tainan--improvement2.png" alt="" title="" style="width:100%"></a>
−
                                        <figcaption>Fig. 4. <i>p</i>-Coumaric acid/OD600 levels of <i>E. coli</i> Nissle with TAL and tyrP in LB with 1mM tyrosine incubated for 48 hours.</figcaption>
+
                                      <figcaption>Fig. 3. Biobrick of <i>soxR-P<sub>soxS</sub>-sfgfp</i></figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
Line 166: Line 128:
 
                         </div>
 
                         </div>
  
−
                         <p>   We compared the TAL constructs containing the native and B0034 ribosome binding sites, (<a href="http://parts.igem.org/Part:BBa_K2997009" target="_blank">BBa_K2997009</a> and <a href="http://parts.igem.org/Part:BBa_K2997010" target="_blank">BBa_K2997010</a>) to determine if <i>p</i>-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig. 4, <a href="http://parts.igem.org/Part:BBa_K2997010" target="_blank">BBa_K2997010</a> is able to produce a higher amount of <i>p</i>-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into <i>p</i>-Coumaric acid can increase by 1.73-fold. Therefore, we have shown that we have improved a previous BioBrick.</p>
+
                         <p>&emsp;&emsp;In this experiment, an overnight culture was prepared, diluted 10 times and transferred into the 96 well plate. The inducer (paraquat-PQ) was added after incubating in 37 °C. Using an ELISA reader, the sfGFP expression level was measured hourly during the 4.5 hours after paraquat induction.</p>
−
                         <p>   For more information, please visit our <a href="https://2019.igem.org/Team:NCKU_Tainan/Results" target="_blank">Results page.</a></p>
+
                  <p>&emsp;&emsp;We compared sfGFP expression levels of <i>soxS</i> promoter with and without the activation of SoxR transcription factor. As shown in Fig. 3, sfGFP expression levels were higher with SoxR than without SoxR. In addition, there was no leakage problem after we added SoxR into our biobrick<sup>[<a href="#ref4">4</a>]</sup> (Fig. 4).</p>
−
                </section>
+
                <div class="container-fluid p-0">
 +
                            <div class="row no-gutters">
 +
                                <div class="col-lg ">
 +
                                    <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
 +
                                        <!-- put link in href & src , width in a can adjust-->
 +
                                        <table>
 +
                <tr>
 +
                <td bgcolor="#FFFAF4"> <a href="https://static.igem.org/mediawiki/parts/6/63/T--NCKU_Tainan--Low_sfGFP_Expression_%28PsoxS%29.png" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/parts/6/63/T--NCKU_Tainan--Low_sfGFP_Expression_%28PsoxS%29.png" alt="" title="" style="width:100%"></a></td>
 +
                <td bgcolor="#FFFAF4"><a href="https://static.igem.org/mediawiki/parts/3/33/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28soxR-PsoxS%29.png" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/parts/3/33/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28soxR-PsoxS%29.png" alt="" title="" style="width:100%"></a></td>
 +
                </tr>
 +
                </table>
 +
                                  <figcaption>Fig. 4. Leakage problem was perfectly solved after the improvement.</figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                            </div>
 +
                         </div>
 +
                  <p>&emsp;&emsp;Please visit <a href="https://2021.igem.org/Team:NCKU_Tainan/Results">Results page</a> for more information.</p>
 +
             
 +
             
 +
              </section>
 
                  
 
                  
 
                 <section class="ref">
 
                 <section class="ref">
Line 174: Line 155:
 
                     <ol>
 
                     <ol>
 
             <!--id name must corespond -->
 
             <!--id name must corespond -->
−
                         <li id="ref1">https://2007.igem.org/wiki/index.php/Edinburgh/Team </li>
+
                         <li id="ref1">Overkamp W, Beilharz K, Detert Oude Weme R, et al. Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging. <i>Applied and Environmental Microbiology</i>. 2013;79(20):6481-6490. doi:10.1128/aem.02033-13</li>
−
                         <li id="ref2">Chen, H., Bjerknes, M., Kumar, R., & Jay, E. (1994). Determination of the optimal aligned spacing between the Shine – Dalgarno sequence and the translation initiation codon of <i>Escherichia coli</i>  mRNAs. <i>Nucleic Acids Research, 22</i>(23), 4953–4957. </li>
+
                      <li id="ref2"><i>Creation, Expression, Purification and Characterization of GFP G4b GFP Expression and Melting Curves.<a href="https://www.biophysik.physik.uni-muenchen.de/teaching/laboratory_courses/gfp_expression/g4b_gfp_expressionenglish_2017.pdf">https://www.biophysik.physik.uni-muenchen.de/teaching/laboratory_courses/gfp_expression/g4b_gfp_expressionenglish_2017.pdf</a> </i></li>
−
                        <li id="ref3">https://2013.igem.org/Team:Uppsala</li>
+
                         <li id="ref3">Baez A, Shiloach J. Escherichia coli avoids high dissolved oxygen stress by activation of SoxRS and manganese-superoxide dismutase. <i>Microbial Cell Factories</i>. 2013;12(1):23. doi:10.1186/1475-2859-12-23</li>
−
                    </ol>
+
                      <li id="ref4">Seo S, Kim D, Szubin R, Palsson Bernhard O. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655. <i>Cell Reports</i>. 2015;12(8):1289-1299. doi:10.1016/j.celrep.2015.07.043</li>
 +
                    <li id="ref5">Part:BBa E0040 - parts.igem.org. Igem.org. Published 2013. Accessed October 16, 2021. <a href="http://parts.igem.org/Part:BBa_E0040">http://parts.igem.org/Part:BBa_E0040</a></li>
 +
                  </ol>
 
                 </section>
 
                 </section>
 
             </main>
 
             </main>

Latest revision as of 03:50, 14 December 2021




Overview

  In our project, the superfolder green fluorescent protein (sfGFP) allows better quantification of promoter strength and sensitivity[1,2]. In the oxidative stress sensing system biobrick, we improved the biobrick BBa_K2610031 from the 2019 iGEM Leiden team by changing GFP (BBa_E0040)[2] into sfGFP(BBa_I746916), which is hypothesized to have a higher expression level than GFP [Fig. 1]. We also added transcription activator SoxR to our biobrick for increased function of the oxidative stress sensing system[3].

Fig.1. Improvement big picture

Results

Disk assay

  Disk assay was used to check the effect of each inducer. The concentration of the different inducers we used are listed below:

Inducer Volume per disk
H2O2 (30%) 10 μl
H2O2 (3%) 10 μl
MD (menadione)(10 mM) 10 μl
DMSO (solvent for MD) 10 μl
PQ (paraquat)(1 mM) 10 μl
MQ (solvent for PQ) 10 μl
Table 1. Oxidative stress inducers

  With sfGFP, the induced system result can be checked under UV light, making it easier to tell the difference between each inducer (Fig. 2).

Fig. 2. The induced result is clear to be seen after the improvement.

Oxidative Stress Assay

Fig. 3. Biobrick of soxR-PsoxS-sfgfp

  In this experiment, an overnight culture was prepared, diluted 10 times and transferred into the 96 well plate. The inducer (paraquat-PQ) was added after incubating in 37 °C. Using an ELISA reader, the sfGFP expression level was measured hourly during the 4.5 hours after paraquat induction.

  We compared sfGFP expression levels of soxS promoter with and without the activation of SoxR transcription factor. As shown in Fig. 3, sfGFP expression levels were higher with SoxR than without SoxR. In addition, there was no leakage problem after we added SoxR into our biobrick[4] (Fig. 4).

Fig. 4. Leakage problem was perfectly solved after the improvement.

  Please visit Results page for more information.

References

  1. Overkamp W, Beilharz K, Detert Oude Weme R, et al. Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging. Applied and Environmental Microbiology. 2013;79(20):6481-6490. doi:10.1128/aem.02033-13
  2. Creation, Expression, Purification and Characterization of GFP G4b GFP Expression and Melting Curves.https://www.biophysik.physik.uni-muenchen.de/teaching/laboratory_courses/gfp_expression/g4b_gfp_expressionenglish_2017.pdf
  3. Baez A, Shiloach J. Escherichia coli avoids high dissolved oxygen stress by activation of SoxRS and manganese-superoxide dismutase. Microbial Cell Factories. 2013;12(1):23. doi:10.1186/1475-2859-12-23
  4. Seo S, Kim D, Szubin R, Palsson Bernhard O. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655. Cell Reports. 2015;12(8):1289-1299. doi:10.1016/j.celrep.2015.07.043
  5. Part:BBa E0040 - parts.igem.org. Igem.org. Published 2013. Accessed October 16, 2021. http://parts.igem.org/Part:BBa_E0040

e-mail : igem.ncku.tainan@gmail.com
phone : 06-2757575