Difference between revisions of "Team:UESTC-China/Improve"

 
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<img src="https://static.igem.org/mediawiki/2018/7/7d/T--UESTC-China--up.png" width="100%">
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<a href="#" class="dropdown-toggle" data-toggle="dropdown">PROJECT</a>
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">PROJECT</a>
 
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<li><a href="https://2018.igem.org/Team:UESTC-China/project_introduction">Introduction</a></li>
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<li><a href="https://2018.igem.org/Team:UESTC-China/Description">Description</a></li>
 
<li><a href="https://2018.igem.org/Team:UESTC-China/Design">Design</a></li>
 
<li><a href="https://2018.igem.org/Team:UESTC-China/Design">Design</a></li>
 
<li><a href="https://2018.igem.org/Team:UESTC-China/Demonstrate">Demonstrate</a></li>
 
<li><a href="https://2018.igem.org/Team:UESTC-China/Demonstrate">Demonstrate</a></li>
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<a href="#" class="dropdown-toggle" data-toggle="dropdown">MODELING</a>
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<a href="#" class="dropdown-toggle" data-toggle="dropdown">MODEL</a>
 
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             This year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. And the part number is BBa_K2617017.The combination of promoter, RBS and signal peptide makes it convenient to use as a Biobrick and gives it the function of extracellular expression.
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             This year, we have improved the previous part <a href="http://parts.igem.org/Part:BBa_J23100" style="color:blue">BBa_J23100</a> by adding RBS and pelB-5D to make it can be used for extracelluar expression. And the part number is <a href="http://parts.igem.org/Part:BBa_K2617017" style="color:blue">BBa_K2617017</a>. The combination of promoter, RBS and signal peptide makes it convenient to use as a Biobrick and gives it the function of extracellular expression.
 
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             For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.
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             For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.The construction of plasmids were as followed.
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      <div class="xstitle">Table 1&nbsp;&nbsp;&nbsp;The introduction of Positive Control and Negative Control</div>
 
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             Experiment Result
 
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             The activity of the extracelluar fractions was detected by the method of PETase. And the results were as followed.
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             For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured(Fig. 1).
 
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         <div class="chatu" style="padding:20px 10%;"><img src="https://static.igem.org/mediawiki/2018/b/bb/T--UESTC-China--improvet1.png" width="100%"></div>
 
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             Figure1.The activity for extracellular fractions of Positive Control and Negative Control
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             Fig. 1 The standard curve of pNP.
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            The activity of the extracelluar fractions was detected by the method of pNPB. And the results were as followed(Fig. 2).
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        <div class="chatu" style="padding:20px 10%;"><img src="https://static.igem.org/mediawiki/2018/8/81/T--UESTC-China--improvet2.png" width="100%"></div>
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            Fig. 2 The activity for extracellular fractions of Positive Control and Negative Control
 
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             Based on our functional determination test results, the improvement that we did has succeeded. As you could see, PETase that carrying our improved part (Figure 1) has much higher activity level than the PETase that carrying promoter J23100 but without pelB-5D (Figure 1). From our experiment result, we could conclude that our improved part indeed gives the promoter J23100 the function of extracellular expression .So, our team has successfully improve the part of J23100 with the addition of RBS and pelB-5D.
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             Based on our functional determination test results, the improvement that we did has succeeded. As you could see, PETase that carrying our improved part (Fig. 2) has much higher activity level than the PETase that carrying promoter BBa_J23100 but without pelB-5D. From our experiment result, we could conclude that our improved part indeed gives the promoter BBa_J23100 the function of extracellular expression. So, our team has successfully improve the part BBa_J23100 with the addition of RBS and pelB-5D.
 
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                 <div class="col-md-12 " style="text-alien: center; vertical-alien: middle; font-size: 127%; text-align: center;">Copyright © 2018 iGEM UESTC_China </div>
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                 <div class="col-md-12 " style="text-alien: center; vertical-alien: middle; font-size: 127%; text-align: center;">Copyright © 2018 iGEM UESTC-China </div>
 
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Latest revision as of 14:55, 17 October 2018

team

IMPROVE PART

  • Part Improvement
    This year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. And the part number is BBa_K2617017. The combination of promoter, RBS and signal peptide makes it convenient to use as a Biobrick and gives it the function of extracellular expression.
    For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.The construction of plasmids were as followed.
    Table 1   The introduction of Positive Control and Negative Control
    No. Vector Vector map Description
    1 Positive Control BBa_J23100-RBS-pelB+5D-PETase-Ter
    2 Negative Control BBa_J23100-RBS-PETase-Ter
  • Experiment Result
    For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured(Fig. 1).
    Fig. 1 The standard curve of pNP.
    The activity of the extracelluar fractions was detected by the method of pNPB. And the results were as followed(Fig. 2).
    Fig. 2 The activity for extracellular fractions of Positive Control and Negative Control
  • Conclusion
    Based on our functional determination test results, the improvement that we did has succeeded. As you could see, PETase that carrying our improved part (Fig. 2) has much higher activity level than the PETase that carrying promoter BBa_J23100 but without pelB-5D. From our experiment result, we could conclude that our improved part indeed gives the promoter BBa_J23100 the function of extracellular expression. So, our team has successfully improve the part BBa_J23100 with the addition of RBS and pelB-5D.
Copyright © 2018 iGEM UESTC-China