Data/hora: 01/06/2024 03:37:07

Provedor de dados: | 5
País: | France
Título: | Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus
Autores: | Birien, Tiphaine; Thiel, Axel; Henneke, Ghislaine; Flament, Didier; Moalic, Yann; Jebbar, Mohamed.
Data: | 2018-02
Ano: | 2018
Palavras-chave: | Archaea; Piezophiles; Hyperthermophiles; Genetics; Gene deletion; Deep sea; Hydrothermal vents.
Resumo: | 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 (&lt;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 &lt;15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., polB and rnhB).
Tipo: | Text
Idioma: | Inglês

Identificador:

https://archimer.ifremer.fr/doc/00425/53679/54523.pdf

DOI:10.3390/genes9020077

https://archimer.ifremer.fr/doc/00425/53679/

Editor: | Mdpi
Formato: | application/pdf
Fonte: | Genes (2073-4425) (Mdpi), 2018-02 , Vol. 9 , N. 2 , P. 77 (13p.)

Fonte:

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/).

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