I am infecting E.Coli(DH5alpha) with lambda EYFP with amber mutation that doesn't allow to lyse cell, but it can multiply inside. (30min on ice and 1,5 hour at 37,MOI=1). When I calculate the number of infected cells using both microscopy ant FACS(The cells have higher signal in YFP channel comparing to the control and I can see the infected cells on snapshots or on dot plots from 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?
It is not clear from your description how you are doing this experiment. Do you have two different stocks of lambda, one from an RM+ strain and one from an RM- strain that you are using to infect DH5, or are you using the same phage stock to infect two different host strains (and if so, what is the other strain? ) . Note that DH5a is R-M+ so should not restrict incoming phage.
Dear Mr Benedik,
I am using one stock of lambda to infect different samples. One is DH5 without RM system, second is DH5 with type II RM system on the plasmid. The strain is the same.
Ok, now it is more clear how you are doing the experiment. One thing that comes to mind is that your results might vary a lot depending upon the Type II system you are using. For example if the there are no recognition sites for the Type II enzyme in EYFP gene, then you might have a situation where the phage genome is cut but the EYFP gene can still express because that segment remains intact. So it might be that your mode of detection with one is more sensitive in liquid can detect a signal from cells where the phage genome is still digested.
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