Hi!
I'm looking for a paper or a protocol of a validated method for testing of genetic stability of bacteria. Does anybody know sth like that? We were looking for the papers on this subject but we could not find anything specific.
Best regards,
Joanna
Hello,
various milecular biological genetical techniques exist but better to chose routine methods which are sensitive and provides reproducible results, but is also cost and time effective.
Commonly used conjugation techniques, colony cross streak, broth mating, combined spread plate, and membrane filtration. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% but in some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the combined spread plate and membrane filtration techniques compared with the broth mating colony cross streak techniques were not statistically significant at the P value less than or equal to 0.05 level.
The stability can be obtained using the conjugation technique by integrating colony cross streak, combined spread plate methods. You will be getting your solution by reviewing the sent file.
Hi Joanna, I think it really depends on what do you want to test and if there is any phenotypic change associated to the genomic change you are interested in. To evaluate genetic stability at the genomic level, some researchers have just propagated individual populations of bacteria and evaluated genetic changes by genome sequencing (eg https://dx.doi.org/10.1186%2F1471-2148-2-12, https://doi.org/10.1099/mic.0.000405, but many others). If you want to evaluate a simpler genomic instability, such as the stability of a mobile genetic element, you might consider tagging the element with an antibiotic resistance cassette and/or a negative selection gene (such as the levansucrase gene sacB). We and other have used this approach to test genetic instability of plasmids and genomic islands; see as examples https://doi.org/10.1186/s13100-019-0149-4 and https://doi.org/10.1371/journal.pgen.1007014. Also, the population dynamics of a genomic island associated with pathogenicity has been studied in a series of very elegant experiments by the group of Dawn Arnold (see for instance https://doi.org/10.1016/j.cub.2005.10.074 and https://doi.org/10.1371/journal.ppat.1002010)
Dear Jesus,
Thank you very much for your response. We have already checked the plasmid stability in our strains, however, I wanted also to look at the losses or rearrangements in the whole genome. So we plan to use a DNA fingerprinting technique, but I was wondering if there are the general rules of eg. kind of media we should use. Should it be a minimal medium? Or just a standard medium used for propagation. How long should we passage the strains to be sure the strain is genetically stable. I will read the papers you sent me, I hope they will put some new light on our problem.
Best regards,
Joanna
Dear Joanna, it is indeed difficult to know what the best experimental conditions will be, and I do not think there is a right answer. Laboratory conditions are always artificial, but they are an easy and good approach. A rich medium will support robust and fast growth, and I think it should not be too different from a minimal medium (but I might be wrong), and temperature could also have an effect on results. You obviously cannot test all possible conditions, and I would suggest to just use parameters normally used to propagate the strains in the lab.
Yes, you are right, we will be not able to test all possible scenarios. Thank you for the discussion.
I think this can help
Measuring genetic stability in bacteria of potential use in genetic engineering.
MV Walter, A Porteous, RJ Seidler
Applied and environmental microbiology 53 (1), 105-109, 1987
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