Hi all,
I am currently testing the fluorescence of a Yersinia bacterial construct which has mRuby2 integrated in the chromosome. This GMO was made a few years ago and worked perfectly fine. However, upon streaking the strains fresh and testing for mRuby2 signal, we observe heterogeneous or no expression of mRuby2. The gene is present as confirmed by PCR, but the bacteria do not fluoresce. Can someone suggest a possible reason for this?
How do you grow them? Is it a dish with agarized solid medium and the separate colonies growing on it? Generally, I would suggest either oxygenation issues (RFP maturation requires strongly aerobic conditions) or contamination of the stock culture. In the latter case, your colonies may have mixed genotype with a pronounced domination of the non-fluorescent phenotype
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