CONSTRUCTION OF THE ECTOINE GENE OPERON FROM VIRGIBACLILLUS SALARIUS USING BASIC ASSEMBLY AND ECTOINE PRODUCTION IN ESCHERICHIA COLI BL21(DE3)

• Main Author: Arman, Zico
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/84411
• Institution: Institut Teknologi Bandung

Ectoine was a cyclic amino acid produced intracellularly by bacteria in response to growing in high-salt environments. This study aimed to construct the ectoine gene operon from Virgibacillus salarius with various design constructs using BASIC assembly, followed by ectoine production in Escherichia coli BL21(DE3). The ectoine gene cluster's construction began with the ectoine gene cluster's isolation using the ectA forward and ectC reverse primer set. The ectoine gene cluster, approximately 2200 bp in length, was successfully isolated and then inserted into plasmids designed with the BASIC assembly method, using different Ribosome Binding Site (RBS) sequences, the constructed plasmid then used to transform into E. coli and generating strain T1 (RBS1-EctABC), T2 (RBS2-EctABC), and T3 (RBS3-EctABC). After all plasmids were assembled, they were transformed into E. coli DH5? for confirmation and subsequently into E. coli BL21(DE3) for ectoine production using 0.1 mM IPTG as the inducer. The initial ectoine production was conducted to observe the effect of IPTG induction and the location of ectoine accumulation during the fermentation process. Strain T3, which had the strongest RBS, was selected as the initial sample for ectoine production, showing an ectoine yield of 0.313 ± 0.021 g/L in the fermentation medium, with more dominant extracellular accumulation. Subsequent production focused on analyzing extracellular ectoine in strains T1, T2, and T3 to observe the influence of different RBS strengths in each transformant colony. Results showed that the T3 sample induced with 0.1 mM IPTG had the highest concentration compared to T1 and T2 colonies, with a concentration of 0.36 ± 0.03 g/L. Further construction was performed on the ectoine gene operon using different RBS for each ectoine gene in one ectoine biosynthesis pathway, namely strain B1 (RBS1-EctA-RBS2-EctB-RBS3-EctC) and B4 (RBS1-EctA-RBS3-EctB-RBS3-EctC). Ectoine concentration analysis was also conducted on strains B1 and B4 to observe the effect of different RBS on each ectoine gene in one ectoine biosynthesis pathway, with the highest results obtained in strain B4 with an ectoine concentration of 0.30 ± 0.06 g/L. However, ectoine production in strain B4 was lower compared to strain T3. Optimization of IPTG concentration from 0 mM, 0.1 mM, 0.5 mM, and 1 mM was then performed on sampel T3 as the sampel with the highest ectoine production in previous experiments. Based on the optimization results, it was found that sampel T3 with 0.1 mM IPTG induction had significantly different ectoine production with an ectoine concentration of 0.37 ± 0.018 g/L (p < 0,05). The results of this study indicated that sampel T3 had the potential for large-scale production, but further optimizations, such as induction compound optimization, glucose content, or the addition of exogenous enzymes to prevent feedback inhibition from ectoine precursors, were still needed.