Research enigma! Cloning in lactobacillus! After transformation I got complete white colonies but after sub culturing the same , It is starting to see red colonies.No idea from where I got those red colonies(my plasmid had RFP but I digested it with R.E and gel confirmation also done). If my restrictions digestion is not proper then I should have red colonies after transformation and sub culturing. perplexed !
Why don't you check a few of the red colonies to see if they carry the RFP gene on the plasmid? It could be that most of your plasmid was correct but some small fraction was original and still has RFP. The other possibility is that the red colonies are not lactobacillus at all but some red contaminant like Serratia which is pretty resistant to most antibiotics.
I placed the plate having red colonies under UV light, Fluorescence was not observed and the plasmid does not have RFP. So serratia like contamination could be a possible reason . Thanks a lot for the suggestions @Prof_Michael.
A good place to check for contamination is elution buffers used for plasmid prep. I've seen the elution buffer of a communal miniprep kit get contaminated with some ampicillin resistant organism before.
If it isn't contamination though, how well do you/your lab know this strain? It's not unheard of for Lactobacilli to be pigmented, and pigmentation could potentially be variable depending on a lot of factors.
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