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A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in Thermococcus kodakarensis, consistently correlated with the TK0664 gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced pyrF by TK0664 on our suicide vector and tested T. barophilus strain sensitivity to 6-MP before and after transformation. Wild-Type (WT) T. barophilus is less sensitive to 6-MP than WT T. kodakarensis, and an increase of cell resistance was achieved after deletion of the T. barophilus TERMP_00517 gene homologous to T. kodakarensis TK0664. Results confirmed the natural resistance of T. barophilus to 6-MP and show that TK0664 can confer sensitivity. This new counterselection system vastly improves genetic manipulations in T. barophilus MP, with a strong decrease in false positives to <15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., polB and rnhB).

Pertenece a

ArchiMer, Institutional Archive of Ifremer (French Research Institute for Exploitation of the Sea)  

Autor(es)

Birien, Tiphaine -  Thiel, Axel -  Henneke, Ghislaine -  Flament, Didier -  Moalic, Yann -  Jebbar, Mohamed - 

Id.: 70993689

Idioma: eng  - 

Versión: 1.0

Estado: Final

Tipo:  application/pdf - 

Palabras clavearchaea - 

Tipo de recurso: Texto Narrativo  -  Publication  -  info:eu-repo/semantics/article  - 

Tipo de Interactividad: Expositivo

Nivel de Interactividad: muy bajo

Audiencia: Estudiante  -  Profesor  -  Autor  - 

Estructura: Atomic

Coste: no

Copyright: sí

: 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Formatos:  application/pdf - 

Requerimientos técnicos:  Browser: Any - 

Relación: [IsBasedOn] Genes (2073-4425) (MDPI AG), 2018-02 , Vol. 9 , N. 2 , P. 77 (1-13)

Fecha de contribución: 14-feb-2018

Contacto:

Localización:
* DOI:10.3390/genes9020077

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