The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r

Abstract

Two oligodeoxyribonucleotides were chemically synthesized and used to specifically mutate the regulatory region of the araBAD operon in Escherichia coli B/r. One oligodeoxyribonucleotide introduced a 3-bp deletion in the araC activator binding site, the other a 3-bp deletion in the CRP-cAMP binding site. The mutations were introduced onto an ara insert cloned in an M13 vector using the synthetic oligodeoxyribonucleotides as primers and the (+) strand of an M13 mp2::ara hybrid phage as a template in an in vitro polymerization reaction. Hybridizations using the original synthetic oligodeoxyribonucleotide as a radioactive probe identified phage containing the desired deletion. The mutant ara inserts were subcloned into a stable plasmid for functional analysis. Transcription studies performed on strains containing the mutant ara plasmids demonstrated that both mutations reduced the amount of araBA mRNA synthesized in the presence of L-arabinose.