Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome.
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).
Abalakina
Elena G
EG
Ajinomoto-Genetika Research Institute, 117545, Moscow, Russian Federation.
Tokmakova
Irina L
IL
Gorshkova
Natalya V
NV
Gak
Evgueni R
ER
Akhverdyan
Valerii Z
VZ
Mashko
Sergey V
SV
Yomantas
Yurgis A V
YA
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
D004251:DNA Transposable Elements