I cloned a toxic protein in E. coli BL21-DE3 and E. coli BL21AI, and the transformation efficiency was very extremely low (1-8 colonies). It is known that basal expression of the cloned genes can occur in these strains in the absence of the inductors. My dilemma is that by inducing expression with IPTG these colonies, I detected the expression of the constructs. So, I have two main questions:
1. How could it be explained that a few bacteria have been able to incorporate the cloned gene taking into account that there may be a basal expression of the toxic protein?
2. Could any genetic variation have occurred either in the cell's host genome or in any regulatory sequence of the vector that could have decreased the basal expression rate, allowing it to tolerate the cloned gene?
It is possible that the clones you obtained acquired a mutation either in the regulatory sequence, or, perhaps, in the origin of replication (leading to a lower copy number, and therefore a lower metabolic burden/toxicity).
I would suggest using glucose in the media when you plate your BL21 transformants to shut down leaky expression. In case of toxic proteins, these create a selection parameter when you collect your colonies. Those that grow in protein presence may have unpredictable mutations in the plasmid template (such as those mentioned earlier). I would also double-check that you did not pick up a mutation in the toxic gene that renders it ineffective.
Good luck!
BL21(DE3) is notoriously prone to leakage of expression in the absence of inducer, particularly if the strain doesn't harbour a second plasmid such as pLysS. This is due in part to the presence of traces of lactose in tryptone, a casein hydrolysate. However, BL21AI affords a much more stringent control and is the host of choice for toxic proteins. In the case of BL21(DE3), there will be a selection for low-expressing variants, e.g. those reducing the number of copies of the plasmid. What is more surprising is that you should be able to induce the BL21AI strain with IPTG, since in this strain synthesis of T7 RNA polymerase required for the expression of the recombinant gene is supposed to be induced by L-arabinose. Does your expression plasmid feature a T7 promoter ? If not, e.g. if it belongs to the pQE series, no need to use strains providing T7 RNA polymerase such as BL21(DE3) or BL21AI . As Ana suggests, adding glucose to the medium will help shut down leakage at any rate, due to catabolite repression.
You may well have isolated a mutant in the cloned gene that no longer is toxic or elsewhere as Ana mentioned. A very easy test is to make a plasmid prep and retransform into fresh cells. If you get high transformation then you have a mutation on your plasmid. If it is very low again then all should be well.
Regardless, Pierre Béguin raises some excellent points about controlling expression.
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