I am currently working on a lactobacilli and trying to construct a deletion mutant of one of its transporter, through cre-lox based deletion system (Lambert et al 2007). But I am facing problem in electroporation, the plasmid is not transforming in the strain i.e. no colony on the antibiotic resistant plate (MRS Cm+). I am following the method of electroporation mentioned in the article but its not working for me.Alternatively I have tried Bio Rad protocol also but that too was unable to give me any positive result in the form of colony. Please suggest and solution.
Dear Deepesh,
The following paper may be helpful for you
Article Improved electroporation and cloning vector system for gram-...
Best wishes
Sunagar Raju , thank you very much for the article. I had gone through it but, we are working on lactobacillus strain and most of the transformation pertaining to lactobacilli (firmicutes) involves incorporation of PEG (polyethylene glycol 1000 or 1500). In this study they have not used any such protocol and neither any lactobacilli strain. But I will still give it an attempt. Thanks once again.
In our experience with lactococcus, after electroporation, we cultured bacteria in a lower concentration of antibiotic and if you obtain colonies then you can transfer them into a standard concentration containing media.
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