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.