RRC ID 4916
Author Chiang CJ, Chen PT, Chao YP.
Title Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.
Journal Biotechnol. Bioeng.
Abstract Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic-resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon-free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site-specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli.
Volume 101(5)
Pages 985-95
Published 2008-12-1
DOI 10.1002/bit.21976
PMID 18553504
MeSH Attachment Sites, Microbiological / genetics* Carotenoids / metabolism Chromosomes / metabolism Cloning, Molecular / methods DNA / genetics DNA / metabolism DNA Nucleotidyltransferases / genetics DNA-Directed RNA Polymerases / genetics DNA-Directed RNA Polymerases / metabolism Escherichia coli / genetics* Escherichia coli / metabolism Farnesyl-Diphosphate Farnesyltransferase / genetics Farnesyltranstransferase / genetics Gene Expression Regulation, Bacterial* Gene Targeting / methods* Genes, Regulator Genetic Enhancement / methods Genetic Markers Genetic Vectors Lycopene Mutagenesis, Insertional / methods* Oxidoreductases / genetics Phosphoenolpyruvate Carboxykinase (ATP) / genetics Plasmids Recombination, Genetic / physiology Replicon / genetics Transferases / genetics Viral Proteins / genetics Viral Proteins / metabolism
IF 3.952
Times Cited 39
Prokaryotes E. coli CV-25(DH5alpha) pINT-ts pAH69 pAH121 pAH130