Here, we outline how to identify hydrogenase enzymes from metagenomic fosmid libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis MR-1 (ΔhyaB) via triparental mating. If a fosmid clone exhibits hydrogen-uptake activity, S. oneidensis’ phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. The screen enables screening of 48 metagenomic fosmid clones in parallel.
CITATION STYLE
Adam-Beyer, N., & Perner, M. (2023). Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes. In Methods in Molecular Biology (Vol. 2555, pp. 91–101). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2795-2_6
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