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Molecular Biology Methods in Streptomyces rimosus, a Producer of Oxytetracycline

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Antimicrobial Therapies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2296))

Abstract

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.

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Acknowledgments

This work was supported by European Union’s Horizon 2020 Research and Innovation Programme TOPCAPI (grant no. 720793) and by the project MISSION founded by the ERA CoBioTech partners, BMBF, MIZS, CDTI, and European Union. This work was also funded by the Slovenian Research Agency (ARRS) (grant no. J4-8226) and ARRS young research grant (grant no. 35220200570). We thank Vasilka Magdevska for figures demonstrating the use of reporter genes in S. rimosus .

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Authors and Affiliations

  1. Chair of Biotechnology, Microbiology and Food Safety, Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

    Lucija Slemc, Špela Pikl, Hrvoje Petković & Martina Avbelj

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  1. Lucija Slemc
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  2. Špela Pikl
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  3. Hrvoje Petković
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  4. Martina Avbelj
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Correspondence to Martina Avbelj .

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  1. INBIOTEC (Instituto de Biotecnología de León), Parque Científico de León, León, Spain

    Carlos Barreiro

  2. Department of Biotechnology, Crystal Pharma A Subsidiary of Albany Molecular Research Inc. (AMRI) Parque Tecnológico de León, León, Spain

    José-Luis Barredo

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Slemc, L., Pikl, Š., Petković, H., Avbelj, M. (2021). Molecular Biology Methods in Streptomyces rimosus, a Producer of Oxytetracycline. In: Barreiro, C., Barredo, JL. (eds) Antimicrobial Therapies. Methods in Molecular Biology, vol 2296. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1358-0_18

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  • DOI: https://doi.org/10.1007/978-1-0716-1358-0_18

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  • Print ISBN: 978-1-0716-1357-3

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