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A comprehensive practical laboratory course on protein engineering: Evolution of a glycine oxidase variant active on the herbicide glyphosate
Biochemistry and Molecular Biology Education ( IF 1.4 ) Pub Date : 2019-08-24 , DOI: 10.1002/bmb.21291
Elena Rosini 1 , Loredano Pollegioni 1
Affiliation  

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. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):689–699, 2019.

中文翻译:

关于蛋白质工程的综合实践实验室课程:对除草剂草甘膦有活性的甘氨酸氧化酶变体的进化

蛋白质工程代表了一种为生物技术的不同领域生成新蛋白质的现代方法。在这里,我们报告了一项为期 8 天的实验室活动,在该活动中,学生生成酶变体来降解除草剂草甘膦。学生在一个重要领域(通过新型工程酶进行生物修复)进行真正的研究实验,并了解分子生物学和蛋白质生物化学实验室中广泛使用的技术。基于甘氨酸(原始底物)和草甘膦与甘氨酸氧化酶活性位点的对接分析,确定了可能参与底物选择性的残基,这些残基将成为位点饱和诱变的目标。每组学生专注于在一个位置生成的库,并根据比色筛选选择最活跃的变体。在大肠杆菌中过表达蛋白质后,纯化选定的甘氨酸氧化酶变体,并评估它们对甘氨酸和草甘膦的动力学特性。然后使用全班鉴定的最佳变体来检测水中的除草剂。在教授的帮助下,学生可以提高技术技能、评估结果的能力、团队合作活动和批判性思维。© 2019 国际生物化学与分子生物学联盟,47(6):689–699,2019。然后使用全班鉴定的最佳变体来检测水中的除草剂。在教授的帮助下,学生可以提高技术技能、评估结果的能力、团队合作活动和批判性思维。© 2019 国际生物化学与分子生物学联盟,47(6):689–699,2019。然后使用全班鉴定的最佳变体来检测水中的除草剂。在教授的帮助下,学生可以提高技术技能、评估结果的能力、团队合作活动和批判性思维。© 2019 国际生物化学与分子生物学联盟,47(6):689–699,2019。
更新日期:2019-08-24
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