#TITLE_ALTERNATIVE#

• Main Author: BAHAR (NIM : 20716040), ZULHAERANA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/24868
• Institution: Institut Teknologi Bandung

Antibiotics selection system in the production of therapeutic proteins can be replaced by lysine auxotrophic selection to avoid the antibiotic resistance spreading. The auxotrophic selection system requires both host cell that has mutated one of gene on its chromosome and plasmid carrying the gene to complement the mutant host cell. In previous study plasmids carrying dapD genes have been constructed for lysine auxotrophic selection system. One way to construct a mutant host cell is the CRISPR/Cas9 method and λ-red homologous recombination. The components involved in this method are Cas9 endonuclease, sgRNA, 20 specific nucleotides (N20dapD for dapD), λ-red recombinase protein, and homologous fragments X1X2. These components present in pCas and pTarget series which are pTargetT-dapD and pTargetF-dapD (for dapD). This research were aimed to construct pTargetF-dapD plasmid, generate lysine auxotroph E. coli BL21(DE3) mutant and confirm the resulting mutant (E. coli BL21(DE3)dapD). This research began by determining N20dapD and homologous fragment X1X2 in silico followed by ordering synthetic gene that carry the fragment on pUC18-N20dapDX1X2. pTargetF-dapD obtained by inserting N20dapD into pTargetF through EcoRI and SpeI restriction sites. Fragment X1X2 was isolated by PCR method. In vivo mutation of dapD gene was performed by electroporating the pCas and induced the expression of λ-red recombinase protein with L-arabinose at final concentration 10 mM before electroporation of E. coli BL21(DE3) with pTarget series. Both plasmids were then removed by curing mechanism using IPTG induction (pTarget) and temperature shifts (pCas). pTargetF-dapD sized 2122 bp was successfully obtained and confirmed by migration, EcoRI digestion, PCR, and sequencing analysis. The X1X2 fragment was isolated and has a 953 bp in size. Both plasmids transformation and curing were successfully performed. Four colonies of lysine auxotroph mutant E. coli gave positive result on genotype analysis by PCR and phenotype analysis using selection medium. In conclusion, this study succeeded in constructing pTargetF-dapD and obtaining the confirmed E. coli BL21(DE3)dapD lysine auxotrophic mutant.