Cloning and Sequencing of Haloacid Dehalogenase Gene from Pseudomonas aeruginosa Local Strain

• Main Author: Dwinta Utami, Lousiana
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32261
• Institution: Institut Teknologi Bandung

Utilization pesticides, herbicides, fungicides, insecticides, solvents and dyes in chemical industries released many halogenated organic compounds to the environment. Poisonous character of these compounds make its long existence and damage the ecosystem. Some microbes known to have an ability to catalyze the dehalogenation reaction, conversing halogenated organic compounds to other compounds with less poisonous. The ability to degrade halogenated organic compounds by Pseudomonas aeruginosa local strain had been studied using monochloroacetate as substrate. This study aimed to clon and sequence the haloacid dehalogenase gene from Pseudomonas aeruginosa local strain. The methods include haloacid dehalogenase gene isolation using PCR approach, cloning and sequencing. Haloacid dehalogenase gene from Pseudomonas aeruginosa local strain has been successfully isolated using specific primer and confirmed with agarose electrophoresis by the existence of paed-d and paed-l fragments of ~750 bp and ~1000-1500 bp respectively. Transformation of these amplified fragments to Eschericia coli TOP 10 is sreened by ampicillin resistence and galactosidase activity. Size screening and colony PCR confirmed paed-d fragment positively inserted in pGEM-T plasmid. Meanwhile, paed-l fragment was confirmed negative. Nucleotide sequence of paed-d fragment shows 99% similarity with haloacid dehalogenase from 25 bacteria of Pseudomonas aeruginosa in GenBank (Accession number: CP006728). Deduced protein level of Paed-l has 100% similarity with haloacid dehalogenase from 81 bacteria of Pseudomonas aeruginosa in GenBank (Accesssion number: WP_003106623.1). Conserved domain analysis showed that the active site of Paed-d located in the centre domain on 120th amino acid of the protein.