8 replies to this topic

[Ic3_88]

I had run agarose gel for E.coli,EcoRI cut. From the gel, all I can see is a smear. I am guessing that there is 0 cut for E.coli, EcoRI cut. Please help!

[bob1]

Why would you assume that?  What did you learn about restriction site frequencies in DNA sequences?  What would you see if the RE cut many times?

[Ic3_88]

I was thinking along two choices. But I really don't know which is the right one.

1) EcoRI was isolated from E.coli. So in order for E.coli to protects its own DNA, every EcoRI site on its DNA was methylated. When agarose gel was runned, very likely supercoiled DNA will be seen.

2) EcoRI site on E.coli DNA was not methylated. Hence, enzyme EcoRI will cut up E.coli DNA many times. This produced a smear effect on agarose gel.

Please enlightened me....

[bob1]

The fact that it is from E. coli should actually tell you that it is used by E. coli - they won't produce something they don't use typically.   EcoR1 is not methylation sensitive at all, it will still cut the DNA!

What would an uncut bacterial genome look like on a gel?

[leelee]

As I understand it, EcoRI won't cut E.coli DNA due to protective methylation. Why would a bacteria produce an endonuclease that will chop up its own DNA? Doesn't make sense.  The use of endonuclease to bacteria is to destroy non-self DNA.

Edited to add- it matters which strain of E.coli your DNA is isolated from though, as there are strains produced which do not have methyltranferases.

Edited by leelee, 13 February 2012 - 08:48 PM.

[Julio-Claudian]

Looking up RE-digested genomic DNA, I found a picture in one of Qiagen's docs showing the digestion of gDNA.

https://picasaweb.go...feat=directlink
______

I'm still learning but from the looks of it, EcoRI will still cut regardless of whether the DNA is methylated or not.

While it's true that it's suicidal to make something "that will chop up its own DNA", could it be different in vivo? bob1, could it be that in vivo, the sensitivity towards methylation is different?

I know it doesn't make much sense how/why the protein could have altered sensitivity in different environment (in vivo vs purified)

The expression of EcoRI for commercial purposes, at least for NEB, was done in the E. coli host.

[bob1]

leelee, on 13 February 2012 - 08:44 PM, said:

As I understand it, EcoRI won't cut E.coli DNA due to protective methylation. Why would a bacteria produce an endonuclease that will chop up its own DNA? Doesn't make sense.  The use of endonuclease to bacteria is to destroy non-self DNA.

As far as I know, EcoR1 is not sensitive to any prokaryotic methylation system.  I would presume that restriction enzymes have some sort of evolutionary advantage due to the fact that they are produced by the bacteria that they come from.  I would hypothesize that they are used to chop invading DNA up and that the genome is protected and repairable, so it doesn't matter to the host that it is producing the RE.

Julio-Claudian, on 13 February 2012 - 09:49 PM, said:

While it's true that it's suicidal to make something "that will chop up its own DNA", could it be different in vivo? bob1, could it be that in vivo, the sensitivity towards methylation is different?

I know it doesn't make much sense how/why the protein could have altered sensitivity in different environment (in vivo vs purified)

I don't think the sensitivity to methylation will be different in different environments, but I don't really know.

[leelee]

So from what I can find, E coli expresses a methyltransferase known as EcoRI adenine DNA methyltransferase, which methylates the host DNA and protects it from the endonuclease.

You can buy it from NEB- http://www.neb.com/n...roductM0211.asp

Edited to add- still looking for a good reference that fully explains this, as all of the ones I've found are talking about non canonical sequences and whilst they refer to the above information (the protection of the host DNA by methylation) I can't find any of the original references, or I don't have access, or they are in Polish

Edited by leelee, 14 February 2012 - 07:40 PM.

[leelee]

By the way, thanks to Ic3_88 for starting this thread, I really like it when I'm reminded that I should probably better understand the background and the basics of the tools that I use in the lab every day!