CONSTRUCTION OF PET16B PLASMID CARRYING TATADCD PROTEIN-ENCODING GENE AND TETRACYCLINE RESISTANCE GENE

• Main Author: Asroriyah, Islakhiatul
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/73765
• Institution: Institut Teknologi Bandung

The Twin Arginine Translocation (TAT) extracellular protein delivery system in bacteria can translocate proteins in the optimal folding conformation. In Bacillus subtilis, there is a TATAdCd protein that was developed for usage in Escherichia coli for the delivery of genetically modified proteins with intracellular folding issues. Antibiotic resistance genes on plasmids are extensively utilized as selection markers in recombinant DNA production. The existence of different antibiotic selection markers allows several plasmids to be inserted into the same host cell. This study aimed to construct recombinant plasmids carrying TATAdCd protein-encoding gene and the addition of tetracycline-resistant gene selection markers. The TATAdCd protein-encoding gene in pTA_TATAdCd WT and the tetracycline-resistant gene in pBR322 was isolated and cloned into pET16b. The constructed plasmids were named as pET16b_TATAdCd, pET16b_TetR, and pET16b_TATAdCd_TetR and confirmed at the DNA and protein levels. At the DNA level, the constructed plasmids were confirmed by migration analysis, restriction analysis, Polymerase Chain Reaction (PCR), and DNA sequencing. At the protein level, the expression of TATAdCd protein was proved by the overproduction of TATAdCd protein in E. coli BL21 (DE3) host cells, and the protein was characterized by SDS-PAGE Tris-Glycine. Expression of tetracycline-resistant genes was demonstrated by growing E. coli DH5? carrying plasmids pET16b_TetR and pET16b_TATAdCd_TetR on Luria Bertani agar medium containing tetracycline. In this study, the expression of TATAd and TATCd proteins could not be observed on Tris-Glycine SDS-PAGE, while the tetracycline-resistant gene was successfully expressed because the recombinant E. coli could grow in tetracycline selection media.