Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome |
| |
Authors: | Elena G. Abalakina Irina L. Tokmakova Natalya V. Gorshkova Evgueni R. Gak Valerii Z. Akhverdyan Sergey V. Mashko Yurgis A. V. Yomantas |
| |
Affiliation: | (1) Ajinomoto-Genetika Research Institute, 117545 Moscow, Russian Federation |
| |
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 α-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). |
| |
Keywords: | Methylotrophs Mini-Mu unit Replicative transposition Amplification Excisable marker |
本文献已被 PubMed SpringerLink 等数据库收录! |