KONSTRUKSI DAN EKSPRESI GEN DNA POLIMERASE MUTAN DELESI BAKTERI TERMOFILIK ISOLAT CIMANGGU DI Escherichia coli

• Main Author: Ekawardhani, Savira
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/1875
• Institution: Institut Teknologi Bandung

Commercially available thermostable DNA Polymerases possess different properties. Hence, studies on novel thermostable DNA Polymerases are still needed. A thermophilic bacterium, closely related to Bacillus thermoleovorans based on its phylogeny, has been isolated from Cimanggu hot spring (pH 5, 70°C). Whole coding sequence of DNA Polymerase 1 gene from the bacterium has been cloned and sequenced. Since structure-function studies on this thermostable DNA Polymerase require mutant enzymes, therefore the aim of this research was to construct deletion (∆) mutants of polymerase gene which was performed by in frame-deletion method using restrictrion enzyme. The over expressed ∆ mutant genes were constructed in the expression vector (pTrxFus) with tryptophan induced system, identified and sequenced. The mutant enzymes were characterized by SDS-PAGE and its polymerase activities. The mutant enzymes which were constructed in this research indicated the following properties : Mutant ∆EcoRl enzyme lost one of its carboxylate triad, i.e Asp653 Mutant ∆Xhol enzyme lost its 5'-3' and 3'-5' exonuclease domain. Mutant ∆RsaI enzyme lost its N-terminal or the 5'-3' exonuclease domain. Qualitative activity assay on mutant ∆EcoRI (89 kDa) crude extract showed that the mutant lost its polymerase activity compared to its wild type. DNA polymerase mutant ∆Xhol gene was not expressed although the plasmid and the sequence of the gene were still unchanged.