Hi there,

T5 promoter will definitely recruit endogenous E. coli RNA polymerase. So it should work in theory. Only experiments will tell which system is actually the best. In theory T7 driven transcription is more efficient than T5.

The pQE vectors from Qiagen utilize the phage T5 promoter that is controlled by two lac operator sequences. The T5 promoter is recognized by the E. coli RNA polymerase and induction is accomplished by IPTG addition to release the Lac repressor from the dual operator sequence. Since pQE vectors do not carry the lacI gene, the host strain must supply an excess of Lac repressor. There are two options exist for LacI supplementation: copropagation of multicopy pREP4 (QIAexpress manual) or use of a strain that carries the lacIq gene. Many K - 12 strains (e.g., JM109) carry the lacIq gene, you can use them if your target protein is less sensitive to protease degradation. Most B strains e.g. C43, do not have lacIq except a few offer LacI overexpression like NEB Express I, which is a BL21 derivative that carries a miniF lacIq gene. You might get your protein expressed in any E. coli strain but the expression is only induction sensitive in these with the expression of lacI.

I really appreciate your answers, I would be very grateful if you could further help me in this issue:

Would we be able to successfully express the protein if we forgo the repressor?

I assume the expression will begin as soon as the plasmids are transformed into the cell in that case. Does the e.coli supply small amounts of the repressor without the lacI gene that we might still need to add IPTG for?

Will expression be too strong and unregulated that the process might be disrupted?

In the case of immidiate expression, what condition should the cells be kept in after transformation and for how long before we can have enough protein to run a gel?

Hi again,

According to C43(DE3) genotype, it bears wt lacI gene and therefore codes for normal level of lacI protein so pQE30 mediated transcription will not be fully locked but it might no be maximal. The possibility of using this system for expression will be dependent on the possible effect of constitutive expression of your protein of interest on the cell viability. The accumulation of protein can't be anticipated so as for any other strategy you have to test/optimize conditions. But the easiest would definitely be to cotransform with plasmid overexpressing lacI to recover a fully inducible system.