Difference between revisions of "Part:BBa K3829009"

 
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<partinfo>BBa_K3829009 short</partinfo>
 
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MHETase is a protein optimized for degradation of MHET. In our experiments, MHETase can degrade MHET (one of the products of PETase degradation) into TPA and EG. As a key part of the surface display system, MHETase achieves our experimental purpose mainly by degrading the MHET.  
 
MHETase is a protein optimized for degradation of MHET. In our experiments, MHETase can degrade MHET (one of the products of PETase degradation) into TPA and EG. As a key part of the surface display system, MHETase achieves our experimental purpose mainly by degrading the MHET.  
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<h3>Characterization</h3>
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<P>On the basis of <a href="https://parts.igem.org/Part:BBa_K3829011">BBa_K3829011</a>, we replaced MHETase with yeGFP to obtain the composite parts BBa_K3829014. The structure was shown in figure 1.</P>
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<img src="https://2021.igem.org/wiki/images/5/5c/T--IvyMaker-China--Lab-51.png" style = "width:70%;">
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<br/><b>Fig.1</b> The structure of the gene circuit.
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<P>In order to evaluate the surface display of MHETase, HPLC was used to detecte the enzyme activity. We set different reaction times, namely 10, 20, 30, 60, and 90 min. The HPLC results showed that the substrate decreased sharply within 60 min, and the degradation was almost complete at 60 min. Besides, MHET could not be detected at 90 minutes, indicating that the display of MHETase was successful.</P>
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<img src="https://2021.igem.org/wiki/images/b/bd/T--IvyMaker-China--Lab-43.png" style = "width:50%;">
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<br/><b>Fig.2</b> Determination of enzyme activity of MHETase.
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<h3>References</h3>
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<p>1.Eisenhaber, Birgit, et al. "A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe." Journal of molecular biology 337.2 (2004): 243-253.</p>
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<p>2.Möller, Steffen, Michael DR Croning, and Rolf Apweiler. "Evaluation of methods for the prediction of membrane spanning regions." Bioinformatics 17.7 (2001): 646-653.</p>
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<p>3.Smith MR, Khera E, Wen F. “Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.</p>
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<p>4.Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.</p>
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<p>5.Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.
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Revision as of 07:10, 21 October 2021


MHETase

MHETase is a protein optimized for degradation of MHET. In our experiments, MHETase can degrade MHET (one of the products of PETase degradation) into TPA and EG. As a key part of the surface display system, MHETase achieves our experimental purpose mainly by degrading the MHET.

Characterization

On the basis of BBa_K3829011, we replaced MHETase with yeGFP to obtain the composite parts BBa_K3829014. The structure was shown in figure 1.


Fig.1 The structure of the gene circuit.

In order to evaluate the surface display of MHETase, HPLC was used to detecte the enzyme activity. We set different reaction times, namely 10, 20, 30, 60, and 90 min. The HPLC results showed that the substrate decreased sharply within 60 min, and the degradation was almost complete at 60 min. Besides, MHET could not be detected at 90 minutes, indicating that the display of MHETase was successful.


Fig.2 Determination of enzyme activity of MHETase.

References

1.Eisenhaber, Birgit, et al. "A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe." Journal of molecular biology 337.2 (2004): 243-253.

2.Möller, Steffen, Michael DR Croning, and Rolf Apweiler. "Evaluation of methods for the prediction of membrane spanning regions." Bioinformatics 17.7 (2001): 646-653.

3.Smith MR, Khera E, Wen F. “Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.

4.Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.

5.Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.

Sequence and Features BBa_K3829009 SequenceAndFeatures