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An improved protocol for electroporation in members of the genus Gordonia

https://doi.org/10.1016/j.mimet.2013.07.025Get rights and content

Highlights

  • The electroporation methods published earlier did not work in Gordonia sp. IITR100.

  • Tween 80 permeabilized the cells and was found to be essential for electroporation.

  • The plasmid isolated from Gordonia sp. IITR100 was found to be structurally stable.

Abstract

Gordonia are high GC gram-positive bacteria that have not yet been exploited well for biotechnological purposes because of the limited genetic tools. Described here is an improved protocol for electroporation, which is useful for several Gordonia species. The maximum transformation efficiency obtained was 2.8 × 104/μg (Gordonia rubropertinctus, Gordonia sp), and 1.7 × 103/μg (Gordonia amarae).

Section snippets

Acknowledgments

The authors would like to thank Dr. Shavandi for the kind gift of plasmid pRSG43 and Dr. Shilpi Sharma of IIT Delhi, India for thoughtful comments. The work was supported by a financial grant from Department of Biotechnology, Government of India.

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Cited by (11)

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    BDS experiments were performed with Gordonia sp. IITR100, which was isolated from petroleum contaminated soil by enrichment culture using 4,6 dimethyl dibenzothiophene as the sulfur source (Gen bank accession number GU084407) [17]. BDS studies, on heavy crude oil and hydrodesulfurized diesel, were performed in shake flask and in bench top bioreactor (5 L Infors AG) equipped with monitoring and control of standard environmental variables.

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    Plasmid from E. coli was isolated using genelute plasmid mini kit (SIGMA, USA) following manufacturer's instructions. Plasmid isolation and electroporation in Gordonia was done following a protocol optimized in our laboratory (Singh and Srivastava, 2013). Restriction enzyme digestion, ligation and transformation were performed using standard methods (Sambrook et al., 1989).

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