Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

Abstract

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.

Construction of expression vectors for protein production in Gluconobacter oxydans

“…Briefly, cells were harvested by centrifugation (4500 × g, 15 min at 4 • C) between an OD 600 nm 0.8 and 1.0, washed three times with ½ volume 1 mM HEPES and resuspended in 250 l HEPES. Glycerol was added to approximately 20% and electrocompetent cells were flash frozen in 65 l aliquots and stored at −70 • C. Electroporation was performed by a modified method of Mostafa et al (2002) with a Gene Pulser II (Bio-Rad, München, Germany) set at 2.0 kV, 25 F and the pulse controller set at 200 with a 1 mm electrode distance.…”

“…Transformation of G. oxydans was done by electroporation essentially as described by Mostafa et al (2002). Briefly, cells were harvested by centrifugation (4500 × g, 15 min at 4 • C) between an OD 600 nm 0.8 and 1.0, washed three times with ½ volume 1 mM HEPES and resuspended in 250 l HEPES.…”

Bacteria of the genus Asaia stably associate with Anopheles stephensi , an Asian malarial mosquito vector

“…stephensi was verified by electroporating Asaia sp. strain SF2.1 with plasmids pHM2 and pHM3, two replicative plasmids in the genera Acetobacter and Gluconobacter closely related to the genus Asaia (22). The plasmids transformed strain SF2.1 with an efficiency of 4.7 10 5 cells⅐g Ϫ1 DNA.…”

Poly(hydroxyalkanoate) Production by Cupriavidus necator from Fatty Waste Can Be Enhanced by phaZ1 Inactivation

“…In a previous work, part of the phaZ1 depolymerase gene was cloned in the suicide plasmid pSUP102, thus obtaining plasmid pSUP102-phaZ1 41 . The gene phZ1 was interrupted in the unique pst1 site by the Km r gene.…”