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

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            <tr><td>No.</td><td> Vector</td><td> Vector map</td><td> Description</td></tr>
 
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            <tr><td>1 </td><td>Positive Control</td><td>  <img src="https://static.igem.org/mediawiki/2018/b/b5/T--UESTC-China--improve1.png" width="100%">  </td><td> BBa_J23100-RBS-pelB+5D-PETase-Ter</td></tr>
 
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            <tr><td>2 </td><td>Negative Control</td><td>  <img src="https://static.igem.org/mediawiki/2018/c/c2/T--UESTC-China--improve2.png" width="100%">  </td><td>  BBa_J23100-RBS-PETase-Ter</td></tr>
 
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             Overview
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             Part Improvement
 
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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. 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 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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            Vector design for the validation of expression system
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             For the verification of function, we decided to use the PETase as our reporter protein. Two plasmids were constructed.
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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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             Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful.
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             <table class="table table-hover">
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                <tr><td>No.</td><td> Vector</td><td> Vector map</td><td> Description</td></tr>
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                <tr><td>1 </td><td>Positive Control</td><td>  <img src="https://static.igem.org/mediawiki/2018/b/b5/T--UESTC-China--improve1.png" width="100%">  </td><td> BBa_J23100-RBS-pelB+5D-PETase-Ter</td></tr>
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                <tr><td>2 </td><td>Negative Control</td><td>  <img src="https://static.igem.org/mediawiki/2018/c/c2/T--UESTC-China--improve2.png" width="100%">  </td><td>  BBa_J23100-RBS-PETase-Ter</td></tr>
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             Validate the extracellular expression of PETase by SDS-PAGE
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             Experiment Result
 
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             To validate the extracellular expression of PETase, the supernatant fractions of LB culture were separated by centrifugation (8000rpm,4℃). Then we used the absorbing column to do the protein concentration. Added 30mL supernatant into absorbing column, and got 1mL even less concentrated supernatant through refrigerated centrifugation(4℃, 4000g，30min).After SDS-PAGE analysis, we found that the supernatant of piGEM2016-001 which carrying pelB-5D showed a target band while no such a band was detected in piGEM2016-002 （FigXX).The result demonstrated that our improved part can increase the extracellular expression of the protein.
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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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        <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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             Detect the activity of PETase
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             Conclusion
 
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             The activity of PETase was determined after expression of PETase in E.coli BL21 (DE3) for 24h.pNP (p-nitrophenol) assay is commonly used method for quantitative detection of lipase activity. The assay is based on the production of pNP which has a maximum absorption at 405 nm. For measuring the activity of PETase, we chose pNPB (p-nitrophenol butyrate) as the substrate which can be hydrolyzed to pNP by PETase. One unit (U) of PETase activity was defined as the amount of PETase that could release 1 mmol pNP from pNPB per minute (Kim et al., 2015). 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 (Figure 8).
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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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            Reference
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Revision as of 04:50, 16 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.
    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
    The activity of the extracelluar fractions was detected by the method of PETase. And the results were as followed.
    Figure1.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 (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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