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Development of a pyrE-based selective system for Thermotoga sp. strain RQ7

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Abstract

To fully unlock the biotechnological potentials of Thermotoga species, this study aimed to expand the genetic toolbox of Thermotoga by developing a new selective system. The developed system was composed of two components: a recipient strain bearing a deletion in its orotate phosphoribosyltransferase gene pyrE and a shuttle vector expressing a heterologous pyrE as the selectable marker. A spontaneous uracil auxotroph, T. sp. strain RQ7-15, was isolated at 70 °C with 2 mg/ml 5-fluoroorotic acid. The mutant carried a 112 bp deletion in pyrE and was a suitable recipient strain. To avoid homologous recombination, the pyrE gene from another thermophilic bacterium Caldicellulosiruptor saccharolyticus was used as the selectable marker. The gene was cloned into two Thermotoga-E. coli shuttle vectors, controlled by different promoters: the promoter of Thermus S-layer protein (P slpA ) in pDH25 and the promoter of the pyrimidine synthesis operon of T. sp. strain RQ7 (P RQ7.pyr ) in pDH28. After being introduced into the mutant strain RQ7-15 through natural transformation, both vectors allowed the host to thrive in a minimal medium. Single colonies of transformants were isolated and confirmed by polymerase chain reactions and restriction digestions. In summary, a pyrE-based selective system has been established in T. sp. strain RQ7.

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Abbreviations

5-FOA:

5-Fluoroorotic acid

bp:

Base pairs

CFU:

Colony forming unit

DNA:

Deoxyribonucleic acid

Kan:

Kanamycin

ORF:

Open reading frame

ori :

Origin of replication

PCR:

Polymerase chain reaction

RQ7:

Thermotoga sp. strain RQ7

RQ7-15:

T. sp. strain RQ7-15, a uracil auxotroph of T. sp. strain RQ7

UMP:

Uridine monophosphate

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Acknowledgements

This work was supported by the BGSU Commercialization Catalyst Award and the BGSU Building Strength Award to ZX.

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Authors and Affiliations

  1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA

    Dongmei Han & Zhaohui Xu

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  1. Dongmei Han
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  2. Zhaohui Xu
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Correspondence to Zhaohui Xu.

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Communicated by L. Huang.

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Han, D., Xu, Z. Development of a pyrE-based selective system for Thermotoga sp. strain RQ7. Extremophiles 21, 297–306 (2017). https://doi.org/10.1007/s00792-016-0902-2

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  • DOI: https://doi.org/10.1007/s00792-016-0902-2

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