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Why a bacterial cell restriction enzyme not cleave foreign gene - (Jun/08/2005 )

Why does a bacterial cell restriction enzyme not cleave any foreign gene inserted in it ?
what happens when we use a synthetic gene?


Maybe you should ask that question in the DNA methylation, chromatin and histone modification discussion group wink.gif


i think that when you do an transformation, part of the DNA is digested. But as you select the bacterias that did retain the plasmid uncutt, you can't measure the proportion of digested DNA...
I'm not sure of this but it's one point of view... blink.gif



I think the answer is that the gene is inserted into a plasmid, which bacteria use commonly to swap genes with other bacteria.

Normally a plasmid would be digested by the restriction enzyme, however, typically plasmids used in molecular biology contain an antibiotic resistance gene, so if you add an antibiotic to your medium, the bacterium is forced to use the plasmid to express the antbiotic resitance gene, hence it will keep the plasmid to stay alive.



i want to add to my previous post that i think that majority of plasmids are not recongnized by bacterias as foreign DNA specie. In fact they got a replication origin and appropriate size that make them comparabe to other plasmids in bacterias. Obviously the fact you add ampicillin in your media ensure to get out the bacterias that kill your plasmid.


My hint earlier was eluding to the fact that DNA becomes mthylated. My understanding is
that Plasmid DNA is not digested like Bacteriophage due to the fact that it, like the host genomic DNA is methylated, this prevents it from being chewed up. I am not 100% sure of this, but am over 90%.


Does it have anything to do with the restriction site? I mean the motif that needs to be recognized by the restriction enzyme to cut.
Maybe the foreign gene does not have the restriction site for the bacterial cell restriction enzyme, therefore it is not cleaved.
I may be wrong.


Plasmids with restriction sites matching the active enzymes of a target organism *do* get cut. The coli REs are gone from most lab strains, so we don't think about this much. There is a race between the methylase, which is protecting the genomic DNA of the cell, and the RE, which is cutting unmethylated DNA. Sometimes, the methylase wins, and the plasmid survives, but this is a low efficiency transformation compared to the ones which take place in the absence of the RE (or the RE site on the plasmid).


Depending on the strain of bateria you use, yes you do get methylation of DNA.

but with conventional lines of bacteria like DH5 and the like, they are usually Dam and Dcm methylase negative so it is not the methylation that is preventing them.

I think fred33's and bob1's answers sounds good!