Kloning DNA Pengkode PhoQ untuk Pengembangan High Throughput Screening System sebagai Usaha Mencari Antibakteri Baru terhadap Salmonella typhi
Daftar Isi:
- Salmonella typhi is a cause of typhoid fever. The major problem in the therapy is drug resistancy. PhoP-PhoQ two-component system is a regulator of many virulent factors of Salmonella typhi used in this research as a potential target of developing new antibacterial agents. The goal of this research is to search new antibacterial agents against Salmonella typhi. The research procedure involved isolating the DNA, optimating phoQ gene amplification, and cloning the gene. These steps were used as a part of a novel screening system for searching new antibacterial agents against Salmonella typhi. S. typhi genome was isolated by using Wizard Isolation Kit, and then PCR were done by using PhoQFWD and PhoQREV as primers. Afterwards, phoQ was cloned by using pGEM-T easy vector. The result was grown in LB agar medium containing ampicillin, IPTG and X-gal, and plasmid with DNA insert was characterized with fast lysis method. The result of DNA isolation is the total cell DNA of Salmonella typhi. PCR was performed in 30 cycles. Denaturation was at 94oC for a minute, annealing at 53oC for a minute, and extension at 72oC for 2 minutes. The result showed a band between 1000 and 1500 bp, close to the expected size 1464 bp. The cloning produced 37 white colonies and 11 blue colonies. After fast lysis, the colonies number 9, 18, 24 and 34 displayed a band with a size larger than that of the empty pGEM-T easy vector and were expected to have been inserted with DNA target. Keywords: Salmonella typhi, PhoP-PhoQ two-component system, DNA isolation, PCR, cloning.