When I am transforming a plasmid in BL21 (DE3) cells, plate is found to contain colonies of different size. Roughly they may be classified as small, medium and large. Are they all equivalent for the purpose of protein expression? If somebody knows please explain it logically.
Some of the big colonies might be fusions of two or more smaller colonies which lie close to each other. It is not the size of the colonies but their appearance that is important. Eliminate those with irregular shapes. Pick those that appear to be perfect circles (to make sure they are single colonies), and "healthy" -- that is, those with well-defined edges and bulge a little bit above the agar surface.
Some variation in colony size is perfectly normal. Not all bacteria in a culture are at the same point of the cell cycle and so not all founder cells will start forming a colony at the same time. Add in variations in physiological state and senescence, environmental injury, competition for nutrients with neighboring colonies on the plate, and you end up with size heterogeneity.
In my experience, in BL21(DE3) at least colony size does not matter for subsequent expression (colony age does, but that variable is the same across the plate).
If you want to be on the safe side, do a Gram stain of one colony from each size class to ensure that they are not bacterial contaminants whose colony morphology is similar to that of BL21(DE3). Also, streak one colony of each size on separate plates. If everyting is OK, I would expect each to produce the same mixture of small, average and large colonies.
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