Adam-Beyer, Nicole and Perner, Mirjam (2023) Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes. In: Metagenomics, Methods and Protocols. , ed. by Streit, Wolfgang R. and Daniel, Rolf. Methods in Molecular Biology, 2555 . Springer, Cham, pp. 91-101, 11 pp. 3rd edition DOI 10.1007/978-1-0716-2795-2_6.

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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.

Document Type:	Book chapter
Funder compliance:	DFG: PE1549-6/1 ; DFG: PE1549-6/3
Keywords:	Metagenome, Functional Metagenomics, Function-based screen, Hydrogenase, Hydrogen-uptake, Conjugation
Research affiliation:	OceanRep > GEOMAR > FB2 Marine Biogeochemistry > FB2-MG Marine Geosystems
Main POF Topic:	PT6: Marine Life
Publisher:	Springer
Date Deposited:	07 Nov 2022 08:47
Last Modified:	19 Jan 2023 11:00
URI:	https://oceanrep.geomar.de/id/eprint/57227

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