Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.
Appl Environ Microbiol
; 77(21): 7595-604, 2011 Nov.
Article
em En
| MEDLINE
| ID: mdl-21908633
ABSTRACT
The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA Bacteriano
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Genoma Bacteriano
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Desulfovibrio vulgaris
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Genômica
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Ensaios de Triagem em Larga Escala
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Genética Microbiana
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article