Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.

Q4 Biochemistry, Genetics and Molecular Biology

Methods in molecular biology Pub Date : 2025-01-01 DOI:10.1007/978-1-0716-4220-7_14

Niels N Oehlmann, Johannes G Rebelein

引用次数: 0

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.

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利用金门克隆技术生成位点饱和突变库并将其转移到广泛的宿主范围质粒上。

蛋白质工程是一种定制酶反应性的成熟方法。一种常用的方法是定向进化，即在实验室中模拟并加速诱变和自然选择过程。在这里，我们介绍一种可靠的方法，通过在含有 pBBR1 复制子的宽宿主范围质粒中进行金门克隆，生成饱和诱变文库。通过生成荚膜罗杆菌铁氮酶基因簇（anfHDGK）的突变文库证明了该方法的适用性，随后对该突变文库进行了筛选，以改进分子氢的形成。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

2.00

自引率

0.00%

发文量

3536

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.