CLONING OF OPEN READING FRAME blaTEM EXTENDED SPECTRUM B-LACTAMASE GENE IN ESCHERICHIA COLI
Bacterial resistance to B-lactam antibiotic can be caused by a long term usage of B-lactam antibiotic itself and its ability to produce B-lactamase enzyme. Extended Spectrum B-lactamase (ESBL) is a member of B-lactamase enzyme produced by negative Gram bacteria which are able to hydrolyze both penic...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/10030 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Bacterial resistance to B-lactam antibiotic can be caused by a long term usage of B-lactam antibiotic itself and its ability to produce B-lactamase enzyme. Extended Spectrum B-lactamase (ESBL) is a member of B-lactamase enzyme produced by negative Gram bacteria which are able to hydrolyze both penicillin and cephalosporin. The objectives of this research were to clone blaTEM open reading frame (ORF) into cloning vector (pGEM-T) and expression vector (pET-32b). The first step was to identify based on the 16SrDNA gene E. coli from RSCM. The second step was isolating both the blaTEM gene and blaTEM open reading frame. The third step was to clone the blaTEM ORF to the vector cloning (pGEM-T) and its characterization. The fourth step was to clone the blaTEM ORF to the expression vector (pET-32b) and its characterization. The result of nucleotide sequencing and comparation to NCBI data bank, E. coli RSCM was confirmed to be E. coli with 99% homology to E.coli BE27. blaTEM gene was successfully isolated from plasmid with 99% homology to the blaTEM-1 gene from E. coli plasmid. The blaTEM ORF with no leader peptide sequence has been successfully isolated by using PCR and ligated into pGEM-T vector. The transformation into E. coli JM109 yielded three transformants; they were two white colonies (S161 and S162) and one blue colony (S8). The result of transformants characterization indicated that one of the transformant, i.e. S162, contained inserted DNA of blaTEM ORF. The transformation into E. coli DH5a yielded three transformants that need to be more characterized. blaTEM ORF from E. coli RSCM was successfully isolated and ligated into pGEM-T vector system. Intracelluler crude extract from Axinella sp. symbionts mixed culture is potentially used as β-lactamase inhibitor for bacteria. The ligation results of blaTEM ORF into expression vector pET-32B need to be more characterized. |
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