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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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