Difference between revisions of "Team:SUIS Shanghai/Contribution"

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        <img src="https://static.igem.org/mediawiki/2019/f/f4/T--SUIS_Shanghai--home_background.jpeg" class="bg" />
  
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<h1>Characterization or Contribution </h1>
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        <div class="main_box" id="main_box">
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<H3>
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Bronze Medal Criterion #5
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            <!--鱼鳞-->
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</H3>
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            <img src="https://static.igem.org/mediawiki/2019/3/3f/T--SUIS_Shanghai--image1.png" class="scale scale1" />
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<p>
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            <img src="https://static.igem.org/mediawiki/2019/3/39/T--SUIS_Shanghai--image2.png" class="scale scale2" />
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<b>Characterization - Standard Tracks:</b> Convince the judges that you have added quantitative experimental characterization data to an existing Part from the Registry of Standard Biological Parts. Clearly document the experimental characterization on the Part's Main Page on the Registry (see the Registry <a href="http://parts.igem.org/Help:Document_Parts">Document Parts page</a> for instructions). The part that you are characterizing must NOT be from a 2019 part number range. It is acceptable to add new data to an already highly characterized part. Please see the <a href="https://2019.igem.org/Measurement/Resources">Measurement Resources page</a> for more information about experimental characterization data. Sample submission is not required.
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<br><br>
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            <img src="https://static.igem.org/mediawiki/2019/8/8d/T--SUIS_Shanghai--image5.png" class="scale scale5" />
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You should list the part(s) you characterized for this medal criterion on this page and include links to the part's Registry pages, but <b>all data must be added to the Part's Main Page on the Registry</b>.
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<b>Contribution - Special Tracks:</b> Document on your team wiki at least one new substantial contribution to the iGEM community that showcases a project related to BioBricks. This contribution should be central to your project and equivalent in difficulty to characterizing a BioBrick Part.
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            <img src="https://static.igem.org/mediawiki/2019/e/ec/T--SUIS_Shanghai--image9.png" class="scale scale9" />
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</DIV>
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            <img src="https://static.igem.org/mediawiki/2019/6/6a/T--SUIS_Shanghai--image11.png" class="scale scale11" />
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 +
 
 +
 
 +
            <div class="title_box">
 +
                <h1>Contribution</h1>
 +
            </div>
 +
 
 +
            <div class="second_box">
 +
 
 +
                <div class="description_box">
 +
 
 +
                    <div class="wrapers">
 +
 
 +
                        <h4>We characterized existing parts which were designed by the 2013 iGEM team from the National Yang Ming University Taiwan (iGEM13_NYMU-Taipei). The parts that we are adding characterization to are the coding sequence – OxyR, a transcription factor protein (BBa_K1104200) and the regulatory sequence – TrxCp (BBa_K1104201), which is a ROS-induced promoter controlled by OxyR. </h4><br />
 +
 
 +
                        <h4>OxyR is activated by ROS (Reactive Oxygen Species) which contains a reactive cysteine residue at position 199 (Cys-199). Upon exposure to a ROS, Cys-199 is oxidized resulting in the formation of an intermolecular disulfide bond between Cys-199 and Cys-208. This causes a conformational change, activating the OxyR transcription factor which can bind to regularly sequences and thus positively regulate some genes. </h4><br />
 +
                        <h4>TrcC promoter is a regulatory sequence for the thioredoxin (TrxC) gene. It contains two Transcription Factor Binding Sites located upstream for OxyR to positively active expression. The OxyR gene then is said to be a positive regulatory gene for the oxidative response as it controls the activation of promoters for the expression of proteins which are induced by treatment with H2O2. </h4><br />
 +
                        <h4>These promoters were used by iGEM13_NYMU-Taipei as a means for detecting infection of bees by the fungus Nosema ceranae. One such promoter is the TrxCp (BBa_K1104201). They also created the part (BBa_K1104241) consiting of the TrxC promoter and the GFP generator (BBa_E0840). These parts have applications for our project as fish are known to produce H2O2 in response to infection by pathogens also. We added our experimental data to each of these parts mentioned above.</h4><br />
 +
 
 +
                        <img src="https://static.igem.org/mediawiki/2019/d/db/T--SUIS_Shanghai--_TrxCp.png" class="textimg">
 +
                        <img src="https://static.igem.org/mediawiki/2019/9/91/T--SUIS_Shanghai--_CompositeTrxCp.png" class="textimg">
 +
 
 +
                        <h4>This construct will express OxyR all the time but downstream genes from TrxC promoter will only be expressed upon binding of active OxyR to the binding regions. OxyR is activated by teh oxidation of a reactive cysteien resiude (Cys-199) by a reactive oxygen species (ROS). </h4><br />
 +
                        <h4>For our characterization we synthesized the circuit used by iGEM13_NYMU-Taipei containing a constitutive promoter expressing OxyR upstream of the TrxC promoter and OxyR binding sites. GFP reporter gene was inserted downstream of the TrxC promoter and thus was said to be under control of ROS. This construct was transformed into Bl21 (DE3) E.coli cells and we performed an assay which measured GFP florescence with treatment with various concentrations of H2O2. </h4><br />
 +
 
 +
                        <table>
 +
                            <tr>
 +
                                <td>H2O2 (mM)</td>
 +
                                <td></td>
 +
                                <td></td>
 +
                                <td></td>
 +
                                <td></td>
 +
                                <td>Average</td>
 +
                                <td>SD</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">5</td>
 +
                                <td>RFV</td>
 +
                                <td>567236</td>
 +
                                <td>575491</td>
 +
                                <td>568431</td>
 +
                                <td>570386</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
 
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.22</td>
 +
                                <td>0.24</td>
 +
                                <td>0.221</td>
 +
                                <td>0.227</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>2578345.45</td>
 +
                                <td>2397879.1</td>
 +
                                <td>2572085.97</td>
 +
                                <td class="bgcoloryellow">2516103.53</td>
 +
                                <td class="bgcoloryellow">83636.2982</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">2.5</td>
 +
                                <td>RFV</td>
 +
                                <td>583944</td>
 +
                                <td>586687</td>
 +
                                <td>580442</td>
 +
                                <td>583691</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.195</td>
 +
                                <td>0.212</td>
 +
                                <td>0.205</td>
 +
                                <td>0.204</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>2994584.61</td>
 +
                                <td>2767391.5</td>
 +
                                <td>2831424.39</td>
 +
                                <td class="bgcoloryellow">2864466.83</td>
 +
                                <td class="bgcoloryellow">95648.7650</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">1</td>
 +
                                <td>RFV</td>
 +
                                <td>945200</td>
 +
                                <td>975187</td>
 +
                                <td>954601</td>
 +
                                <td>958329.333</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.31</td>
 +
                                <td>0.312</td>
 +
                                <td>0.323</td>
 +
                                <td>0.315</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>3049032.25</td>
 +
                                <td>3125599.3</td>
 +
                                <td>2955421.05</td>
 +
                                <td class="bgcoloryellow">3043350.89</td>
 +
                                <td class="bgcoloryellow">69591.0552</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">0.1</td>
 +
                                <td>RFV</td>
 +
                                <td>450025</td>
 +
                                <td>449077</td>
 +
                                <td>448871</td>
 +
                                <td>449324.333</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.302</td>
 +
                                <td>0.299</td>
 +
                                <td>0.294</td>
 +
                                <td>0.29833333</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>1490149.00</td>
 +
                                <td>1501929.7</td>
 +
                                <td>1526772.10</td>
 +
                                <td class="bgcoloryellow">1506283.62</td>
 +
                                <td class="bgcoloryellow">15264.9923</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">0.01</td>
 +
                                <td>RFV</td>
 +
                                <td>303234</td>
 +
                                <td>307976</td>
 +
                                <td>308938</td>
 +
                                <td>306716</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.341</td>
 +
                                <td>0.338</td>
 +
                                <td>0.338</td>
 +
                                <td>0.339</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>889249.266</td>
 +
                                <td>911171.59</td>
 +
                                <td>914017.751</td>
 +
                                <td class="bgcoloryellow">904812.872</td>
 +
                                <td class="bgcoloryellow">11066.3002</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">0</td>
 +
                                <td>RFV</td>
 +
                                <td>180294</td>
 +
                                <td>183250</td>
 +
                                <td>183005</td>
 +
                                <td>182183</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.183</td>
 +
                                <td>0.184</td>
 +
                                <td>0.19</td>
 +
                                <td>0.18566666</td>
 +
                                <td></td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td>985213.114</td>
 +
                                <td>995923.91</td>
 +
                                <td>963184.210</td>
 +
                                <td class="bgcoloryellow">981440.412</td>
 +
                                <td class="bgcoloryellow">13629.5509</td>
 +
                            </tr>
 +
 
 +
                        </table>
 +
 
 +
                        <div class="height50"></div>
 +
 
 +
                        <table>
 +
                            <tr>
 +
                                <td colspan="2">H2O2 (mM)</td>
 +
                                <td>Average</td>
 +
                                <td>SD</td>
 +
                            </tr>
 +
 
 +
                            <tr>
 +
                                <td rowspan="3">5</td>
 +
                                <td>RFV</td>
 +
                                <td>488242</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.648</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">753459.8765</td>
 +
                                <td class="bgcoloryellow">49720</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">2.5</td>
 +
                                <td>RFV</td>
 +
                                <td>456371</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.585</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">780121.3675</td>
 +
                                <td class="bgcoloryellow">38567</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">1</td>
 +
                                <td>RFV</td>
 +
                                <td>499338.5</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.612</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">815912.5817</td>
 +
                                <td class="bgcoloryellow">30909</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">0.1</td>
 +
                                <td>RFV</td>
 +
                                <td>481699</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.573</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">840661.4311</td>
 +
                                <td class="bgcoloryellow">36491</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">0.01</td>
 +
                                <td>RFV</td>
 +
                                <td>468741</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.586</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">799899.3174</td>
 +
                                <td class="bgcoloryellow">49183</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td rowspan="3">0</td>
 +
                                <td>RFV</td>
 +
                                <td>473121.5</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>OD600</td>
 +
                                <td>0.599</td>
 +
                                <td></td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>RFV/OD</td>
 +
                                <td class="bgcoloryellow">789852.2538</td>
 +
                                <td class="bgcoloryellow">67900</td>
 +
                            </tr>
 +
 
 +
                        </table>
 +
 
 +
                        <img src="https://static.igem.org/mediawiki/2019/9/95/T--SUIS_Shanghai--Experiment_OxyR_Characterization_Result.png" class="textimg">
 +
 
 +
                        <h4>This is the relative fluorescence value (RFV)/OD600, an indication of promoter performance, of sample A and a control group under different H2O2 concentration. Similar to the trend of the original result from team NYMU-Taipei, the RFV/OD600 value tend to increase as the concentration of H2O2 increase, despite the gradual decreasing fluorescence value after the concentration exceed 1mM. Accelerating protein degradation of GFP might be a possible explanation for this distinction. Protein degradabilities is said to increase along with peroxide concenration. </h4><br />
 +
                        <h3>References:</h3>
 +
                        <h4>Cheolju Lee, Soon Mi Lee, Partha Mukhopadhyay, Seung Jun Kim, Sang Chul Lee, Woo-Sung Ahn, . . . Seong Eon Ryu. (2004). Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nature Structural & Molecular Biology,11(12), 1179-85.</h4><br />
 +
 
 +
                        <h4>Fredrik Åslund, Ming Zheng, Jon Beckwith, & Gisela Storz. (1999). Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status. Proceedings of the National Academy of Sciences of the United States of America,96(11), 6161-6165.</h4><br />
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Revision as of 12:19, 21 October 2019


Contribution

We characterized existing parts which were designed by the 2013 iGEM team from the National Yang Ming University Taiwan (iGEM13_NYMU-Taipei). The parts that we are adding characterization to are the coding sequence – OxyR, a transcription factor protein (BBa_K1104200) and the regulatory sequence – TrxCp (BBa_K1104201), which is a ROS-induced promoter controlled by OxyR.


OxyR is activated by ROS (Reactive Oxygen Species) which contains a reactive cysteine residue at position 199 (Cys-199). Upon exposure to a ROS, Cys-199 is oxidized resulting in the formation of an intermolecular disulfide bond between Cys-199 and Cys-208. This causes a conformational change, activating the OxyR transcription factor which can bind to regularly sequences and thus positively regulate some genes.


TrcC promoter is a regulatory sequence for the thioredoxin (TrxC) gene. It contains two Transcription Factor Binding Sites located upstream for OxyR to positively active expression. The OxyR gene then is said to be a positive regulatory gene for the oxidative response as it controls the activation of promoters for the expression of proteins which are induced by treatment with H2O2.


These promoters were used by iGEM13_NYMU-Taipei as a means for detecting infection of bees by the fungus Nosema ceranae. One such promoter is the TrxCp (BBa_K1104201). They also created the part (BBa_K1104241) consiting of the TrxC promoter and the GFP generator (BBa_E0840). These parts have applications for our project as fish are known to produce H2O2 in response to infection by pathogens also. We added our experimental data to each of these parts mentioned above.


This construct will express OxyR all the time but downstream genes from TrxC promoter will only be expressed upon binding of active OxyR to the binding regions. OxyR is activated by teh oxidation of a reactive cysteien resiude (Cys-199) by a reactive oxygen species (ROS).


For our characterization we synthesized the circuit used by iGEM13_NYMU-Taipei containing a constitutive promoter expressing OxyR upstream of the TrxC promoter and OxyR binding sites. GFP reporter gene was inserted downstream of the TrxC promoter and thus was said to be under control of ROS. This construct was transformed into Bl21 (DE3) E.coli cells and we performed an assay which measured GFP florescence with treatment with various concentrations of H2O2.


H2O2 (mM) Average SD
5 RFV 567236 575491 568431 570386
OD600 0.22 0.24 0.221 0.227
RFV/OD 2578345.45 2397879.1 2572085.97 2516103.53 83636.2982
2.5 RFV 583944 586687 580442 583691
OD600 0.195 0.212 0.205 0.204
RFV/OD 2994584.61 2767391.5 2831424.39 2864466.83 95648.7650
1 RFV 945200 975187 954601 958329.333
OD600 0.31 0.312 0.323 0.315
RFV/OD 3049032.25 3125599.3 2955421.05 3043350.89 69591.0552
0.1 RFV 450025 449077 448871 449324.333
OD600 0.302 0.299 0.294 0.29833333
RFV/OD 1490149.00 1501929.7 1526772.10 1506283.62 15264.9923
0.01 RFV 303234 307976 308938 306716
OD600 0.341 0.338 0.338 0.339
RFV/OD 889249.266 911171.59 914017.751 904812.872 11066.3002
0 RFV 180294 183250 183005 182183
OD600 0.183 0.184 0.19 0.18566666
RFV/OD 985213.114 995923.91 963184.210 981440.412 13629.5509
H2O2 (mM) Average SD
5 RFV 488242
OD600 0.648
RFV/OD 753459.8765 49720
2.5 RFV 456371
OD600 0.585
RFV/OD 780121.3675 38567
1 RFV 499338.5
OD600 0.612
RFV/OD 815912.5817 30909
0.1 RFV 481699
OD600 0.573
RFV/OD 840661.4311 36491
0.01 RFV 468741
OD600 0.586
RFV/OD 799899.3174 49183
0 RFV 473121.5
OD600 0.599
RFV/OD 789852.2538 67900

This is the relative fluorescence value (RFV)/OD600, an indication of promoter performance, of sample A and a control group under different H2O2 concentration. Similar to the trend of the original result from team NYMU-Taipei, the RFV/OD600 value tend to increase as the concentration of H2O2 increase, despite the gradual decreasing fluorescence value after the concentration exceed 1mM. Accelerating protein degradation of GFP might be a possible explanation for this distinction. Protein degradabilities is said to increase along with peroxide concenration.


References:

Cheolju Lee, Soon Mi Lee, Partha Mukhopadhyay, Seung Jun Kim, Sang Chul Lee, Woo-Sung Ahn, . . . Seong Eon Ryu. (2004). Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nature Structural & Molecular Biology,11(12), 1179-85.


Fredrik Åslund, Ming Zheng, Jon Beckwith, & Gisela Storz. (1999). Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status. Proceedings of the National Academy of Sciences of the United States of America,96(11), 6161-6165.


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