Difference between revisions of "Part:BBa K3829008"
| Line 67: | Line 67: | ||
<h3>References</h3> | <h3>References</h3> | ||
| − | <p>1.Reference: Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667. | + | <p>1.Reference: Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667.</p> |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 02:37, 6 October 2022
PETase
PETase is a protein optimized for degradation of PET. In our experiments, it will be expressed on the surface of the host-Candida Tropicalis. As a key part of the surface display system, PETase achieves our experimental purpose mainly by degrading the PET.
Characterization
PETase was involved in the structure of BBa_K3829013, which was shown in figure 1.
Fig.1 The structure of the gene circuit.
The overall enzyme activity of PETase was measured. Since PETase could also catalyze substrates into p-nitrophenol, a crude test was carried out to show the enzyme activity at different temperatures. The results showed that PET-4609 and 5105 performed better than the wild type ATCC20336 and cytPET (Figure 2).
Fig.2 Determination of enzyme activity of PETase.
It was acknowledged that PETase was able to degrade PET into TPA and MHET. According to the HPLC detection results, the product content of the degraded PET powder of each strain were plotted (Figure 3). It was demonstrated that target products were not detected in the group of ATCC 20336. Compared with the control strain, degradation products were obviously presented in the strain PET-4609 and PET-5105, indicating PETase was displayed on the surface of Candida tropicalis with high enzyme activity. However, several products were detected in the strain cytPET which should not be detected theoretically. It was speculated that a part of PETase was released due to the lysis of cells.
In conclusion, these results demonstrated that the surface display system was indeed able to degrade PET, which was consistent with the previous results.
Fig.3 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.
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.
Improvement: IvyMaker-China 2022 iGEM Team
Characterization
PETase is a key enzyme for degrading plastics. This year we have improved the enzyme, which is a contribution to BBa_K3829008. According to the latest report, we have synthesized Fast-PETase. Fast-PETase have been reported to have higher enzyme activity. The results showed that the enzyme activity of Fast-PETase was indeed higher than that of wild-type PETase.
We also measured the effectiveness of FAST-PETase more directly by testing its effect with degrading PET powder. Specifically, we took the following steps. First, we collected an appropriate amount of cultivated strains and washed it three times with 50 mM glycine-NaOH (pH 9.0-10) buffer. Second, the bacteria were incubated with 1 mL buffer containing 50 mM glycine-NaOH (pH 9.0) and 10 mg PET powder at 30℃ with a speed of 900 r/min. Third, the reaction was terminated by diluting the aqueous solution with 18 mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by heat treatment (85°C, 10 min). Fourth, the supernatant obtained by centrifugation (15,000 × g, 10 min) was analyzed by HPLC. The result shown in the figure below reflected a significantly larger concentration of degraded PET and MHET with FAST-PETase than wild PETase, consistent under different OD conditions.
< img src="https://static.igem.wiki/teams/4122/wiki/parts/05-comparison-of-enzyme-activities-of-fast-and-wild-petase.png" style = "width:70%;">Fig.2 Comparison of enzyme activities of fast and wild PETase.
< img src="Comparison of FAST-PETase and wild PETase with HPLC analysis of degraded PET" style = "width:40%;float:middle;margin left:10px;margin top:30px;">
Fig.3 Comparison of FAST-PETase and wild PETase with HPLC analysis of degraded PET.
References
1.Reference: Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667.
Sequence and Features