I have E. coli which produce Galectin 3. We were given a stock from another lab, which we made a plasmid stock from. It is under an IPTG promoter, but this is leaky and So there is a base layer of Gal-3 produced.
We’ve had trouble since however when I transformed fresh bacteria with the plasmid.
The bacteria grows on Ampicillin plates, and still produces Gal-3 in a leaky way, but when I upscale the process to larger volumes with IPTG induction Gal-3 isn’t produced in large amounts. We’ve had the plasmid checked and it still has the Amp resistance, Gal-3 and IPTG promoter. I've tried a time course of when to start IPTG induction with no differences seen. Gal-3 is normally purified with lactose beads after E. coli homogenisation, and so isn’t tagged.
Does anyone have any advice on what might be wrong?
Kind regards,
Ross
Current Protein and Peptide Science, 2006, 7, 47-56 47
Expression of Highly Toxic Genes in E. coli: Special Strategies and Genetic
Tools
F. Saïda, M. Uzan, B. Odaert and F. Bontems
Article Expression of Highly Toxic Genes in E. coli: Special Strateg...
While this is rather overkill, it is a good review on methods to get tight control over expression.
Thanks, well the ampicillin and IPTG are both fresh stocks, so I’m sure they should be fine. Likewise the sequencing done in the purified plasmid showed that was okay.
I‘m not sure what the original bacterial strain was, but we were using BL21s. So I’ll get in touch with them to make sure its the same/ no additives were put in.
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