Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes

Nicole Adam-Beyer; Mirjam Perner

doi:10.1007/978-1-0716-2795-2_6

Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes

Methods Mol Biol. 2023:2555:91-101. doi: 10.1007/978-1-0716-2795-2_6.

Authors

Nicole Adam-Beyer¹, Mirjam Perner²

Affiliations

¹ Geomicrobiology, Department of Marine Biogeochemistry GEOMAR Helmholtz Centre for Ocean Research Kiel, Wischhofstraße, Kiel, Germany. nadam@geomar.de.
² Geomicrobiology, Department of Marine Biogeochemistry GEOMAR Helmholtz Centre for Ocean Research Kiel, Wischhofstraße, Kiel, Germany.

PMID: 36306080
DOI: 10.1007/978-1-0716-2795-2_6

Abstract

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.

Keywords: Conjugation; Function-based screen; Functional Metagenomics; Hydrogen-uptake; Hydrogenase; Metagenome.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli / genetics
Gene Library
Hydrogen
Hydrogenase* / genetics
Metagenome
Metagenomics

Substances

Hydrogenase
Hydrogen