Our lab has a binary vector for plants. The vector has two resistance antibiotic genes (Kanamysin and Hygromysin). That vector was cloned into E.coli DH5a since more than 7 years ago. Recently, we have problems to construct that vector with our interested gene. I think about some mutation or homologous recombination in that vector. I just want to know, is it possible the mutation occurs in that vector? and exactly, how fast that mutation will be?
The mutation rate or evolution rate of any plasmid or chromosome is difficult to measure. But also, the average rate does not really apply to any individual molecule or clone. As with automobile accidents for an analogy, we may be able to measure that the rate of accidents is exactly 2.35 fatalities per million miles driven, we cannot tell you if you will have an accident today. Also, somewhat similar to auto accidents, there are thousands of types of mutations, from single base substitutions in silent sites, to gene deletions and major rearrangements of DNA.
With any biological materials such as E. coli strains or the plasmids in them, it can be very important to go back to freezer stock frozen down at the time of the first experiment, rather than culturing and passaging them continuously.
Halo Brian Foley,
Thank you for your explain.
But, I need to know. usually how long we can put in the bacterial in stock frozen? I mean in glycerol solution. Because, some of my case, the bacterial could not growth after several years in stock frozen.
If cells (mammalian, plant, bacterial or other) are correctly frozen down and remain stored in liquid nitrogen or a freezer with continuous power, no freeze-thaw cycles, they should remain viable and unchanged (no mutations etc) for many centuries. It is always recommended to take one of the frozen vials out soon after freezing (while other samples of the stock are still growing in the lab) to make sure they survived the freeze/thaw cycle with high viability. Bacterial cells are much easier to freeze, much less "fragile" than mammalian cells, so once you have the procedure proven to work of a given species (like E. coli) you probably don't need to do this test every time you freeze down a set of vials.
Freeze/thaw cycles can kill cells, even with incomplete thawing such as will happen in most frost-free electric freezers. The ice crystals will change shapes and penetrate cell walls. Liquid nitrogen, at −196 °C, or a -50 Celcius freezer, is very different than -10 degrees Celcius.
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