Cloning and Characterization of the β-Lactamase Gene in Escherichia Coli NTUH 9501-1

Several clinical isolates of Escherichia coli from National Taiwan Univer-sity Hospital (HTUH) were shown to be ampicillin-resistant and NTUH 9501-1 strain showed the highest activity level of β-lactamase when penicillin was used as substrate, We used an improved SDS-lysis method to isolate large R plasmid from this strain. The purified R plasmid DNA was digested with vari-ous restriction eudonucleases and tested by Southern blot analysis. A restrict-ion fragment (3254-3954, 692bp) of Pbr322 bearing the TEM-1 β-lactamase gene was used as probe after digestion with Dra I. We used again plaque hydridization to detect recombinant M13 DNA carrying cloned β-lactamase gene. We had determined the nucleotide sequence of the ampicllin resistant gene of recombinant plasmid (Ptw2) that encodes a TEM-1 β-lactamase. Nu-cleotide sequencing revealed that the TEM-1 β-lactamase gene of Ptw2 is almost identical with that of Pbr322, but there were 3bp changes (position 226, C→T; position 436, C→T; position 604, G→T). Besides, in order to determine whether a β-lactamase gene in Ptw2 is active in transformed E.coli cells, three biochemical methods namely iodometric method, hydroxylam-ine reaction and fluorescent product formation were employed, and the results demonstrated unequivocally that the β-lactamase gene is fully active expres-sion in the transformed cells.