3.2 Characterization of the effect of different fore‑cistron sequences on the performance of bicistronic T7 expression systems
The expression plasmid was constructed in E. coli JM109 and later transferred into E. coli BL21, a more efficient strain for recombinant protein expression. To evaluate the performance of different fore‑cistron sequences in the bicistronic T7 expression system, EGFP fluorescence intensity was measured and standardized with OD600 after 24 h of cultivation in MWP. The EGFP expression level in pET28-EGFP with a monocistronic T7 served as the control. The results in Fig. 2A showed that extensive fluorescence intensity of EGFP was observed in bicistronic T7 systems, with 10 of the 16 vectors showing enhanced EGFP expression levels compared to the control. These findings indicated that the fore-cistron sequence has a significant effect on the expression of the target gene, which can enhance or weaken their expression. Finally, the four strongest vectors, pET28-HT5, pET28-HT8, pET28-HT12Y, and pET28-HP11Y, were selected, and their EGFP expression levels increased by 68%, 65%, 76%, and 80%, respectively.
To investigate the underlying reason for the increased EGFP expression level in the bicistronic T7 system, we tested the EGFP transcription level of the top four strongest BCD vectors and calculated their translation efficiency. Unlike the generally improved translation efficiency we previously observed in C. glutamicum ,[19] different effects of fore-cistron sequences were observed across the four vectors (Fig. 2B ). For example, pET28-HT5-EGFP exhibited increased EGFP expression due to enhanced translation efficiency, while pET28-HP11Y-EGFP showed decreased translation efficiency and improved EGFP fluorescence intensity attributed solely to increased transcriptional level. The reasons behind this deserve further exploration.