{"title":"Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.","authors":"Niels N Oehlmann, Johannes G Rebelein","doi":"10.1007/978-1-0716-4220-7_14","DOIUrl":null,"url":null,"abstract":"<p><p>Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2850 ","pages":"251-264"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-4220-7_14","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
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Abstract
Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.
期刊介绍：
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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