Attempted construction of an arabinose-inducible pBAD28 recombinant expression vector containing acrD, an Escherichia coli aminoglycoside efflux porin

Abstract

The construction of pBAD28- acr D, an arabinose-inducible expression vector for AcrD, an aminoglycoside efflux pump, represents a considerable opportunity for investigation of the function of this membrane protein in an experimental setting. In this study, an attempt was made at the construction of this vector by PCR amplification of acr D from the genome of E. coli MG1655, isolation of the expression vector pBAD28 from E. coli DH5α, followed by restriction digestion using HindIII and XbaI, then T4 ligation of the digestion products. Following ® transformation of the ligation reactions into chemically competent E. cloni 5-alpha strain cells and characterization of putative transformants, it was determined that a successful pBAD28- acr D expression vector failed to be constructed, and the transformants were primarily empty pBAD28 vectors.