I am infecting E.Coli(DH5alpha) with lambda EYFP(30min on ice and 1,5 hour at 37,MOI=1). When I calculate the number of infected cells using both microscopy ant FACS I see that in samples with RM system there is only 2 times less infected cells then in the samples without RM. On the plates the same RM system showed protection(using regular phage lambda) at least 2 levels of magnitude. Could somebody suggest what could be the reason of this?
Usually RM systems do show many orders of magnitude (probably more than 2) reduction of non-modified phages such as lambda. However without knowing the details of your experiment it is hard to determine what the problem is. What strain(s) were the lambda lysate grown on and what are your RM+ and RM- comparison hosts? Also how are you analyzing infected cells? The RM system won't prevent infection but rather degrades the infecting DNA post-infection.
Both LE392 and DH5alpha are R-M+ strains, so I would not expect that you see any sort of restriction in this experiment. It may be that your phage is not lysing the DH5alpha cells for other reasons depending upon the genotype of the phage. For example if the phage is carrying the Sam7 mutation it would not form plaques on DH5. However more likely is that DH5 is a recA mutant and lambda forms tiny plaques on recA mutants and often does not show visible lysis. You might think about using a different strain.
Yes the phage doesn't lyse Dh5alpha because it's genotype, but it could infect it and multiply in it(this I see by detecting the increase in fluorescence). But what I can't understand is why the effect of RM system increases the protection only by 2 times when I infect the liquid culture of cells(in falcon), while on the plates with regular phage I can see the increase of the protection on 2 levels of magnitude.
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