Protein engineering represents a modern approach to generate novel proteins for the different fields of biotechnology. Here, we report about an 8-day laboratory activity in which students generate enzyme variants to degrade the herbicide glyphosate. The students conduct a true research experiment in an important field (bioremediation by novel, engineered enzymes) and are introduced to widely used techniques in molecular biology and protein biochemistry laboratories. Based on a docking analysis of glycine (the original substrate) and of glyphosate into the active site of glycine oxidase, residues putatively involved in substrate selectivity are identified that will become the target of site-saturation mutagenesis. Each group of students focuses on the library generated at one position and selects the most active variant based on colorimetric screening. Following protein overexpression in Escherichia coli, the selected glycine oxidase variants are purified and their kinetic properties on glycine and glyphosate assessed. The best variant identified by the whole class is then used for detecting the herbicide in water. With the help of the professor, students can improve technical skills, ability to evaluate results, team work activity, and critical thinking. (c) 2019 International Union of Biochemistry and Molecular Biology, 2019.

A comprehensive practical laboratory course on protein engineering: Evolution of a glycine oxidase variant active on the herbicide glyphosate

Rosini E.
;
Pollegioni L.
2019

Abstract

Protein engineering represents a modern approach to generate novel proteins for the different fields of biotechnology. Here, we report about an 8-day laboratory activity in which students generate enzyme variants to degrade the herbicide glyphosate. The students conduct a true research experiment in an important field (bioremediation by novel, engineered enzymes) and are introduced to widely used techniques in molecular biology and protein biochemistry laboratories. Based on a docking analysis of glycine (the original substrate) and of glyphosate into the active site of glycine oxidase, residues putatively involved in substrate selectivity are identified that will become the target of site-saturation mutagenesis. Each group of students focuses on the library generated at one position and selects the most active variant based on colorimetric screening. Following protein overexpression in Escherichia coli, the selected glycine oxidase variants are purified and their kinetic properties on glycine and glyphosate assessed. The best variant identified by the whole class is then used for detecting the herbicide in water. With the help of the professor, students can improve technical skills, ability to evaluate results, team work activity, and critical thinking. (c) 2019 International Union of Biochemistry and Molecular Biology, 2019.
http://iubmb.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)1539-3429/
active learning/upper-level laboratory activity; colorimetric screening; kinetic properties; Site-saturation mutagenesis
Rosini, E.; Pollegioni, L.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/2081919
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