I am working on several unrelated enzymes that produce toxic products. All genes have been cloned into T7 promoter containing expression vectors, yet when transforming to non-expression hosts such as DH5alpha or XL10, the resulting clones still grow very slow. Transformation to BL21(DE3) results in even slower growth, and addition of 5% glucose only helps to a small degree.
For most of the genes, use of BL21(DE3)pLysS solves the growth problem. However for one of the genes, cloning to DH5alpha results in clones that grow too slow to obtain plasmid, and direct cloning to pLysS results in extremely small colonies that only appear after a few days. The problem should not be related to the gene sequence because I have no problem cloning an inactive mutant.
Any ideas to optimize the cloning and protein expression?
Hi Christopher,
It's really strange for that the plasmid contains toxic gene still cause slow growth rate in the non-expression hosts. However, the growth rate and the size of colony of transformed bacteria are depend on plasmid. But I think that the extremely small colonies appeared few days later is just contaminant bacteria.
Check the digested profile of the plasmid, which hard to be replicated, and confirm there is no deficiency on the anti-antibiotics gene.
If you concerned that the toxic gene is leaked and causing the slow growth rate, try an inducible system.
Hope this help.
I would recommend using Lemo21(DE3) strain from NEB which allows tunable expression of T7 by adding L-rhamnose to the expression culture. This would potentially reduce the growth inhibitory effects of the toxic peptides. I have personally used the strain to express few very toxic genes and the tuning of T7 significantly helped to manage the cytotoxicity of the genes.
I hope, that the attached publication will be useful for you.
Jarek
All of the answers are helpful - thank you. To start with, I am going to try out Soran's suggestion to use Lemo21(DE3).
hello... My publication work is identical to yours.I would like to suggest first clone your gene in pbsks plasmid. You can get desired recombinant after 14hours. KS plasmid is nice one compare to commercial clonning vector which suppose to be high chance of false clone.If you need more helpor protocol,you can send e mail.
For some reason I had better luck cloning the toxic gene in an expression vector using Jm109. I wonder if there is any advantage of Jm109 over DH5alpha in this regard, or if the successful cloning was due to other factors.
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