Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans.
Methods Mol Biol
; 881: 99-136, 2012.
Article
en En
| MEDLINE
| ID: mdl-22639212
ABSTRACT
Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Thiobacillus
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos