title: Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome.
abstract: A phage Mu-driven two-plasmid system for DNA integration in Escherichia coli genome has been adjusted for Methylophilus methylotrophus. Constructed helper plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors MuA and MuB. Integrative plasmids that are only replicated in E. coli could be mobilized to M. methylotrophus and contained mini-Mu unit with a short terminus of Mu DNA, Mu-attL/R. Mini-Mu unit was integrated in the M. methylotrophus genome via mobilization of the integrative plasmid to the cells carrying the helper in conditions of thermo-induced expression of MuA and MuB. In this system, mini-Mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the M. methylotrophus chromosome in the presence of helper plasmid. A kan-gene flanked by FRT sites was inserted in one of the mini-Mu units, and it could be readily excised by yeast FLP recombinase that is encoded by the designed plasmid. The multiple Mu-driven gene insertion was carried out by integration of the Bacillus amyloliquefaciens alpha-amylase gene followed by curing the KmR marker before integration of the second mini-Mu unit with Pseudomonas putida xylE gene encoding catechol 2,3-dioxygenase (C23O).
authors.affiliation: Ajinomoto-Genetika Research Institute, 117545, Moscow, Russian Federation.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Abalakina
authors.forename: Irina L
authors.initials: IL
authors.lastname: Tokmakova
authors.forename: Natalya V
authors.initials: NV
authors.lastname: Gorshkova
authors.forename: Evgueni R
authors.initials: ER
authors.lastname: Gak
authors.forename: Valerii Z
authors.initials: VZ
authors.lastname: Akhverdyan
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Mashko
authors.forename: Yurgis A V
authors.initials: YA
authors.lastname: Yomantas
mesh_terms: D010583:Bacteriophage mu / Q000235:genetics*; D002876:Chromosomes, Bacterial / Q000235:genetics; D004251:DNA Transposable Elements; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial; D020630:Methylophilus methylotrophus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D004251:DNA Transposable Elements