GENERATED OF LYSINE AUXOTROPHIC Escherichia coli DH5?BY CRISPR-Cas9

• Main Author: Nur Ramdani, Anisa
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40491
• Institution: Institut Teknologi Bandung

Antibiotic-based selection systems in recombinant DNA technology have been restricted by regulatory agency due to safety reason. The selection system can be changed with other selection systems such as the auxotroph selection system. Auxotroph selection system requires mutant host cells that unable to synthesize an organic substance which necessary for its viability. The method of directed mutagenesis towards host cell chromosomes is Clustered Regular Interspaced Short Palindromic Repeats-CRISPR related to protein 9 (CRISPR-Cas9) and homologous recombination ?-red. CRISPR-Cas9 requires Cas9 endonuclease, sgRNA, 20 specific target gene nucleotides (for dapD, N20dapD), ?-red recombinase proteins, and H1H2 homologous fragments. The components have owned by plasmid pRed-cas9-recAdapD that intended to mutate the dapD gene in E. coli DH5?which designed and constructed in this study. The aimed of this study was to construct E. coli DH5? (?dapD) auxotrophic strain for lysine and confirm it. This study was initiated by determining the N20dapD and the H1H2 homologous fragments followed by plasmid constructing pRed-cas9-recA-dapD by replacing the N20 sequence and H1H2 homologous fragments on the plasmid pRed-cas9-recA-?poxB. Plasmid pRed-cas9- recA-dapD then confirm by migration and cutting analysis. pRed-cas9-recA-dapD transformed into E. coli DH5?by electroporation. E. coli DH5?culture which has a plasmid inducted by L-arabinose with a final concentration was 2 g /L. Confirmation of mutations was carried out by colony Polymerase chain reaction (PCR), then the plasmid were removed from the mutated colonies through curing treatment with incubation at 37°C. Four mutant E. coli DH5?colonies of the auxotrophic lysine strain gave positive results on genotypic analysis through PCR and phenotype through media selection. In this study, E. coli DH5?(?dapD) mutant auxotrophic lysine strains have been obtained and confirmed by phenotypically and genotypically testing. In further studies, this mutant strain can be used for selection of plasmid systems carrying dapD genes such as pCAD-sod-cer-dapD.