Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina

Gao, H., Murugesan, B., Hoßbach, J., Evans, S. K., Stark, W. M. and Smith, M. C.M. (2019) Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina. BMC Biotechnology, 19, 32. (doi: 10.1186/s12896-019-0521-y) (PMID:31164159) (PMCID:PMC6549336)

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Abstract

Background: Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Results: Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. Conclusions: The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.

Item Type:Articles
Keywords:Amycolatopsis, integrating vectors, R4 integrase, rare actinomycetes, TG1 integrase.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Stark, Professor Marshall
Authors: Gao, H., Murugesan, B., Hoßbach, J., Evans, S. K., Stark, W. M., and Smith, M. C.M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:BMC Biotechnology
Publisher:BioMed Central
ISSN:1472-6750
ISSN (Online):1472-6750
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in BMC Biotechnology 19: 32
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
589142A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1RI MOLECULAR CELL & SYSTEMS BIOLOGY