Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker.
Sci Rep
; 5: 8712, 2015 Mar 04.
Article
en En
| MEDLINE
| ID: mdl-25736821
ABSTRACT
Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13â
kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Regiones Promotoras Genéticas
/
Genoma Bacteriano
/
Ingeniería Metabólica
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Sci Rep
Año:
2015
Tipo del documento:
Article