Vol. 12, Special Issue 7 (2023)

The objective of the present study was to investigate the effect of recombinant pestiviral-based Classical Swine Fever Virus rescue plasmid (pCSFV) propagation in Stbl3 chemically competent cells. We hypothesized that transforming E. coli Stbl3 strain with recombinant pestiviral plasmid may somehow affect its growth pattern. To test this hypothesis, we used a novel RNA-polymerase-II driven pestiviral based pCSFV plasmid as base plasmid for incorporation of GFP reporter gene. The resulting plasmid was transformed into E. coli Stbl3 competent cells through the heat-shock method. Plasmid isolated from positively transformed colonies when cultivated in SB (Super Broth) showed optimum bacterial growth at 72 hours of post-cultivation, indicating the metabolic burden inside bacterial host, due to recombinant pestiviral plasmid hampering bacterial growth. Bacterial cells hosting pestiviral rescue plasmid took 12 to 96 hours to attained OD600 0.065 to 1.45. In contrast, normally bacterial cells attain

~1.6 OD600 within 16 hours of cultivation. Plasmid isolation and restriction analysis at 12-hour intervals till 96 hours showed that up to 36 hours, no plasmid could be isolated since no bacterial growth was observed; at 48 hours, significantly less concentration plasmid was obtained. About 2-3 µg/µl plasmid was isolated at 72-96 hours of cultivation. Isolated plasmid showed an expected 16.384 bp plasmid when run with an X-large DNA ladder in 0.8% agarose gel. Characterization by restriction enzyme digestion with StuI enzyme at 72-96 hours showed 6501 bp, 5230 bp, 2730 bp, 1583 bp, and 340 bp digested products, and PCR amplified product confirmed GFP gene insertion into the pCSFV rescue plasmid showing 872 bp product in 1.5% agarose gel electrophoresis. Overall, the present study sheds light on the propagation of recombinant pestiviral-based plasmids in E. coli Stbl3 cells and highlights the potential benefits of using this strain to maintain such plasmids in industrial-scale bacterial cultures.