Part:BBa_K3829014
P-ss-MHETase-V5tag-Anchor protein 5105-T
The composite Part is used to express MHETase. SS secretes MHETase out of the cell, while anchor protein 5105 anchor it on the cell surface. This part is based onBBa_K3829011, replacing GFP with MHETase.
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| None |