| Popis: |
The T7 expression system is commonly used to obtain high yields of heterologous proteins in Escherichia coli. Still, laboratories empirically optimize its use, frequently with little information for rationally choosing the change of settings in each case. Here, we monitored the expression of green fluorescent protein (GFP) as a function of growth in three strains, with and without additional LacI repressor and IPTG added at different times. BL21(DE3) strain showed the best yield; however, its inability to repress T7 harmed the culture unless additional LacI was provided. The BLR(DE3) and MG1655 strains suppressed T7 leaking by themselves but differed in the outcomes of additional LacI. While LacI overexpression improved BLR’s growth and yield, it severely harmed the MG1655 culture. In all cases, early induction with IPTG offered a better outcome than adding it too late. MG1655 was least sensitive to this timing thanks to its capability of keeping a high growth rate for longer. Notably, the GFP production rate was linear to growth rates throughout the culture, meaning that the ribosomal fraction allocated to GFP production was constant at all the analyzed time points. This observation is consistent with a general, time-resolved regulation function and supports the interpretation of our data through a proteome allocation model (PAM). A simple PAM simulated correctly the carbon overflow triggered by heterologous overexpression, indicating that if any anabolic fraction is added to compete for proteome allocation, the rate of precursors consumption will decrease, thus leading to said overflow. This model proposes that a LacI promiscuous repression activity can explain its observed opposite effects on BLR(DE3) and MG1655 strains. Through different levels of downregulation, the carbon fixating proteome fraction would reach a state closer or farther from optimum in each host. |
