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Title 

Scarless chromosomal gene knockout methods

Authors 

Bong Hyun SungJ H LeeS C Kim

Publisher 

Humana Press

Issue Date 

2011

Citation 

Methods in Molecular Biology, vol. 765, no. 0, pp. 43-54

Keywords 

λ-Red systemI-SceIpREDIRhamnose and arabinose induction systemsacB/sucroseScarless deletion

Abstract 

An improved and rapid genomic engineering method has been developed for the construction of -custom-designed microorganisms by scarless chromosomal gene knockouts. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via scarless deletion of selected genomic regions. The deletion process is mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (1) an arabinose-inducible promoter that drives expression of λ-RED recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (2) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which accomplishes deletion of the introduced marker by double-strand breakage - mediated intramolecular recombination. This genomic deletion is performed simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette are nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

ISSN 

1064-3745

Link 

http://dx.doi.org/10.1007/978-1-61779-197-0_3

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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