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Appl Microbiol Biotechnol. 2011 Aug;91(4):857-71. doi: 10.1007/s00253-011-3416-y. Epub 2011 Jun 23.

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria--mini review.

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1
Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation.

Abstract

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with "excisable" E elements in methylotrophic cells.

PMID:
21698377
PMCID:
PMC3145075
DOI:
10.1007/s00253-011-3416-y
[Indexed for MEDLINE]
Free PMC Article
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