Dear colleagues,
I am wondering whether the size of an individual e.coli colony matters as to how much plasmid it produces? Would larger colonies give higher plasmid yield, or would smaller colonies give more plasmid yield due to toxicity of the plasmid product on the bacteria?
Thanks,
Kind regards,
Maria
There is not an easy answer to your question because it depends upon the situation. If you have a plasmid where most of the colonies are small, and you see some large ones, it could be the large one are mutants of some sort that remove some toxicity issue.
You can also get colony size variants that are host cell related and have nothing much to do with the plasmids, for example some big fat colonies are mucoid variants of E. coli.
In general I try to pick the most average looking colony on a plate and stay away from the either much smaller or much larger rare ones.
But size is probably not going to dictate plasmid yield very much but it might predict growth rate of the culture.
Michael J. Benedik thanks for your suggestion, apparently, the size of colony matters in some cases, indeed. The small colonies seem to contain largest number of plasmid, while biggest are either empty, or are mutants. In attached image, the colony PCR of 0.4 kb fragment shows that smallest colony (colony 5) has the most plasmid.
I don't think you can conclude that there is more plasmid in the smaller colonies by using a colony PCR. Standard PCR is not a quantitative assay and once above a threshold amount the template is usually in excess.
Often with colony PCR you get inhibition by the gunk from the colonies, so it may be that the smaller colonies have less gunk (due to fewer cells). I've had quite a few instances where I got no signal from colony PCR, but then if I diluted the template about 5-10 fold I would get signal.
But I think the right conclusion is to not worry too much about it.
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