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An efficient recombination system for chromosome engineering in Escherichia coli
- Source :
- Proceedings of the National Academy of Sciences of the United States of America. 97(11)
- Publication Year :
- 2000
-
Abstract
- A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective λ prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo . The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective λ prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.
- Subjects :
- DNA, Bacterial
Chromosome engineering
DNA, Recombinant
Biology
medicine.disease_cause
Polymerase Chain Reaction
Recombineering
chemistry.chemical_compound
Viral Proteins
Plasmid
Operon
medicine
Escherichia coli
Prophage
Genetics
Recombination, Genetic
Bacterial artificial chromosome
Multidisciplinary
Circular bacterial chromosome
Temperature
Defective Viruses
Chromosomes, Bacterial
Biological Sciences
Bacteriophage lambda
DNA-Binding Proteins
Exodeoxyribonucleases
chemistry
Oligodeoxyribonucleotides
Genes, Bacterial
Transformation, Bacterial
Genetic Engineering
DNA
Plasmids
Subjects
Details
- ISSN :
- 00278424
- Volume :
- 97
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Accession number :
- edsair.doi.dedup.....6f066c31da4fa99f96ff54c320f41446