S. Ghanem
Cite this Article: S. Ghanem (2011). Cloning and functional analysis of the neomycin phosphotransferase gene (nptII) in Escherichia coli. Genet. Mol. Res. 10(3): 1445-1454. 10.4238/vol10-3gmr1334

Genet. Mol. Res. 10 (3): 1445-1454 (2011)
Published July 19, 2011
DOI: 10.4238/vol10-3gmr1334

About the Authors:

S. Ghanem
Corresponding author: S. Ghanem
E-mail: samah.ghanem@laposte.net

ABSTRACT

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5a ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5a; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.

Key words: Escherichia coli; Neomycin phosphotransferase gene (nptII); Homologous recombination gene (recA); Aminoglycoside resistance.