CONSTRUCTION OF pTargetT_AraC PLASMID FOR DEVELOPMENT OF araC GENE DELETED Escherichia coli BL21 (DE3) MUTANT VIA CRISPR-Cas9

• Main Author: Ayu Fajar (10414021), Putri
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30020
• Institution: Institut Teknologi Bandung

<p align="justify">One of the efforts made in order to discover novel antiviral and antibiotic compound is the development of dimer-based drug screening system (“Dimer-based Screening System”/DBSS). The system employs chimeric protein made of dimerization domain of protein of a pathogen fused to DNA binding domain of AraC repressor to regulate expression of reporter gene EmGFP. The system uses Escherichia coli BL21 (DE3) cell as its host, but this host has limitation due to the basal expression of native AraC protein which can interfere with DBSS system. Therefore, mutagenesis to delete araC gene is needed to establish a reliable host through CRISPR-Cas9 genome editing process. This research aims to construct pTargetT_AraC plasmid which is needed for the deletion of araC gene. Construction is done by substituting fragment flanked by SpeI and EcoRI restriction sites in pTargetF backbone with synthetic gene fragment containing araC-targetting N20, sgRNA scaffold, terminator, and homolog DNA donor. The construction begins with in silico study to design N20 and homolog donor. Next, pTargetF and pUC18 containing synthetic gene are cut using SpeI and EcoRI consecutively. Linear backbone and synthetic gene are purified and ligated; and consequently transformed to E. coli dh5&#945;. Transformants are screened through colony PCR using primers pTargetT_Forw (5’-TGATGCGGTATTTTCTCCTT–3’) and pTargetT_Rev (5’-CGAGTAGGGATAACAGGGTA-3’). Based on colony PCR, 11 of 12 colonies contain prospective pTargetT_AraC plasmid. Three of them are sequenced and it has been found that one of the colonies has plasmid with sequence identity 100%. Therefore, pTargetT_AraC has been successfully constructed and confirmed. This plasmid has potential to be used in CRISPR-Cas9 genome editing to delete araC gene from E. coli BL21 (DE3) genome.<p align="justify">