Random screening of promoters from Escherichia coli and classification based on the promoter strength

Abstract

Five hundred fifty DNA fragments 100-500 base pairs in length were cloned from total chromosomal DNA of Escherichia coli, each capable of promoting the synthesis of beta-lactamase when inserted upstream of the ampC structural gene without its own promoter in a promoter-probe plasmid. All clones in this library of putative promoters were classified based on the level of resistance to ampicillin, which ranged from 10 to more than 1,500 micrograms/ml. Most of the higher levels of drug resistance (more than 1,000 micrograms/ml) were due not only to an increase in gene expression but also to an increase in plasmid copy number. The DNA fragments which produced the highest level of drug resistance all mapped at 5.7 min on the E. coli chromosome and shared the same nucleotide sequence. In these fragments, a strong promoter was found, which carries an up stream AT-rich sequence in addition to -35 and -10 signals of the promoter consensus.