BIOINFORMATICS ANALYSIS FOR ISOLATING A GENE ENCODING SORTASE F FROM ROSSELLOMOREA AQUIMARIS MKSC 6.2

• Main Author: William Sugiarta, Nicholas
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/81365
• Institution: Institut Teknologi Bandung

Sortases have a physiological role of expressing proteins in the cell wall of Gram-positive bacteria. Expressed proteins have various functions, especially in bacterial pathogenesis and virulence. There are various types of sortases (from A-F), each with unique characteristics and physiological roles. Sortase A from Staphylococcus aureus, commonly regarded as the organism’s house-keeping protein, has been extensively studied and gave insight to the inner workings of sortase. The catalytic core of sortase comprises of a catalytic triad made from cysteine, histidine, and arginine, making sortase a cysteine protease. Sortase F is the least understood sortase, but it is thought to have similar mechanisms and functions to sortase A with differing independency towards Ca2+. Rossellomorea aquimaris, a Gram-positive bacterium, has both sortase D and sortase F, the former of which had been studied. The physiological role of sortase F is not well-understood. This research aims to give structural insight of sortase F from R. aquimaris MKSC 6.2 using bioinformatics analysis, and to develop a method of isolating and amplifying its gene (srtF). Our results show that R. aquimaris has two types of sortase F, named sortase F 5T_A and 5T_B. Both proteins have different signal peptides and sortase 5T_B has an additional N-terminal domain, which gave insight into their respective physiological roles. Further bioinformatics analyses are used to design a set of primers based on conserved sortase F catalytic domain. Gene amplification using this set of primers resulted in a 222 bp fragments, which are then ligated to pGEM-T Easy cloning factor. The fragments are then sequenced and identified. Electropherogram analysis does not show expected results. Degenerated primers based on conserved catalytic domains yielded no result, so we designed a new pair of primers based on the whole sequence of srtF 5T_B.