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

Niels N Oehlmann; Johannes G Rebelein

doi:10.1007/978-1-0716-4220-7_14

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

Methods Mol Biol. 2025:2850:251-264. doi: 10.1007/978-1-0716-4220-7_14.

Authors

Niels N Oehlmann¹, Johannes G Rebelein^{2

3}

Affiliations

¹ Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
² Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. [email protected].
³ Center for Synthetic Microbiology (SYNMIKRO), Philipps-University Marburg, Marburg, Germany. [email protected].

PMID: 39363076
DOI: 10.1007/978-1-0716-4220-7_14

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.

Keywords: Broad host range plasmid; Directed evolution; Golden Gate cloning; H2 evolution; Iron nitrogenase; Library generation; Rhodobacter capsulatus; Site saturation mutagenesis.

MeSH terms

Cloning, Molecular* / methods
Directed Molecular Evolution / methods
Gene Library*
Host Specificity / genetics
Multigene Family
Mutagenesis / genetics
Mutagenesis, Site-Directed / methods
Plasmids* / genetics
Rhodobacter capsulatus / genetics