Isolation and Characterization of str Promoter from Bacteria Escherichia coli DH5α using Reporter Gene AmilCP (Acropora millepora)

Abstract

In heat stress conditions, E. coli will produce EF-Tu protein abundantly under the regulation of str promoter. The str promoter from E. coli is known to have sequences that are recognized by two sigma factors (σ70 and σS). The aim of this research was to isolate and characterize str promoter from E. coli DH5a using reporter gene AmilCP. The str promoter from E. coli DH5α (289 bp) had been isolated by Colony PCR using specific primers. Bioinformatics analysis of str promoter showed high conserved areas (100%) for some kinds of E. coli strains. The str promoter was constructed in pSB1C3 vector by modifying OEPC to regulate reporter gene AmilCP. Qualitatively, the ability of str promoter constructs in regulating AmilCP gene was observed as purple-blue colonies of E. coli. Quantitative analysis was performed by measuring cell extract of E. coli at a wavelength of 588 nm after treated at different temperatures (4, 24, 37, 45, 50, 55 and 60 °C) for 1 hour. The ability of str promo ter to express reporter gene AmilCP was compared to one powerful promoter which is the lacUV5 promoter. The results show that both promoters are functional promoters and can be induced by temperature (50 °C).