A colleague of mine has some diffuculty with his RT-PCR experiment. He started analyzing the single failure-prone steps telling that he has restreaked the strain of interest due to some growth impairment. Since he restreaked a single colony on a new plate growth has recovered but the Real-Time PCR shows low expression of the gene of interest and even the Housekeeping gene. Hence, I wondered and have heard already before that repeated restreaking of a single colony may lead to genetic instability, is this correct? Many thanks for an expert advice or publication regarding this matter. Thank you!
I might need a little more to go on here but for now I'm going to assume that when you mention 'strain' you are talking about a microbial strain. If this is the case then your thoughts on genomic instability might very well be the reason.
If this is a microbial strain then the question has to turn to antibiotic selection pressure. If this is a recombinant strain then it must be grown in the presence of the antibiotic it is resistant to at all times. If not the small number of cells without the selection gene will out compete those with it and the gene(s) of interest will be 'lost' (really they will just be a minority of the population). This effect is what is commonly know in lab jargon as 'losing a plasmid' or a cell 'spitting out a plasmid'. Ampicillin is notorious for this. In fact ampicillin can be degraded (beta-lactam ring broken) by heat/light and so cultures grown with it should not go longer than 12 hrs, especially if they are grown in the presence of light.
Ok, so if it is not a recombinant microbial strain using antibiotic resistance then your genomic instability thought still might be the reason, but this depends on the strain and if it has a lot of insertion elements/sequences. If this is the issue then a new plate will have to be restruck from the original glycerol/lyophilized stock almost every time to ensure success.
There might be a few other possibilities but I'll need to know a little more.
Hopefully the antibiotic issue is the problem as that is the easiest to manage.
Hope the above helps your colleague.
Hi James,
I appreciate your comprehensive response and in fact it is about microcin producer E. coli G3/10 that my colleague investigates for its toxic protein expression under certain conditions. I believe this microbial strain has no antibiotics resistance gene but I need to find out first. Thank you for the moment.
It is also not uncommon for strains to become attenuated when grown under laboratory conditions; those select generally for fast growth, and have plenty of nutrients. Many antibiotics will be repressed under those conditions. I am not familiar with regulation of microcin production, but I would not be surprised if it is catabolite repressed and requires growth on limiting medium. Repeated streaking of the same colony is never a good idea if you want reproducible results, you will basically conduct an in-lab evolution experiment.
Hi Wiep, Hi Traci,
I´d like to thank you very much for these valuable answers which I´ll discuss with my colleague soon. Best wishes!
I agree with Traci. The repeated streaking results in the decreased efficiency of microorganisms. It can be better observed in enzyme activities and antibiotic resistance test.
Hi Arti,
Thank you for your response, I´ll consider it with my individual experiments and will talk about this general problem with my colleague. Greetings
In addition to the answers above, all of which I have also seen, don't forget there is often an unconscious bias when selecting which colony to pick and restreak. We often tend to pick the bigger or healthier looking one, but that introduces a bias towards fast growth on that specific medium which may not be relevant to the organisms or phenotype you wish to study.
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