6 replies to this topic

[Chris22]

 

 Hello all,

 

I am hoping to get some input (methods, suggested assays or reagents etc) on a bacterial methylation issue.

 

I am currently working with a gram negative Bacteroides species.

This organism is particularly resistant to transformation, which has only been achieved for one not-so closely related species. A summer student of mine recently attempted to make a genomic library of this organism and noticed poor digestion with typical enzymes used. 

 

I suspect extensive methylation with MTase/Restriction systems are playing a role in both issues. Attached image shows 12 hour digestion with 5U/ug gDNA with four enzymes which all recognize GATC sequence, but differ in methylation sensitivities. (I should note that DpnI actually requires Dam methylation of GATC to cut)

 

Would I be correct to assume based on this result that both CpG methylation and Dam methylation are present and inhibiting digestion? 

 

Also, I am assuming that heavy methylation (12 hours at 5U should completely digest 1ug of DNA!?) likely suggests strong restriction endonuclease activity = no transformation success.

 

Has anyone worked with a similar issue? if so....

 

Is SMRT sequencing the only way to characterize specific methylation patterns in gDNA?

 

Is there a method (low expense?) to isolate endogenous methylases from a bacterial strain?

 

And lastly....as most E.coli are Dam+, theoretically if I methylated plasmid DNA from E.coli with a CpG methylase (NEB, M.ssl), this should block the RE (at least the ones involved in GATC sequence) and improve transformation??? 

 

Hopefully someone else has overcome similar issues in the past and might have a few sly tricks..

 

Thanks!!

 

Chris22 

 

Attached Files

•  Slide Bioforum.pdf   112.54KB   485 downloads

Edited by Chris22, 28 July 2015 - 08:55 AM.

[bob1]

CpG methylation is only found in eukaryotes as far as I know, so this shouldn't be a problem for the REs.

 

Were you expecting to have a single band in the digest, or was this supposed to be a lower MW smear for the library prep? 

 

Most E coli strains have a both Dam and Dcm methylation functional, wouldn't this be enough to prevent digestion in most cases? 

 

What methods of transformation have you attempted?

[phage434]

Here's a useful trick: PCR out your DNA insert using a dNTP mix which replaces dCTP with 5-me dCTP. You may have to fiddle with cycling conditions, but your DNA will then be resistant to the restriction enzymes present in your construct. You can also eliminate all GATC sequences in your constructs (I do that with my organism).

If you need a plasmid, amplify and ligate (your ligation region and primers can't contain a GATC).

 

DAM methylation of E. coli won't do this, since it methylates the A, not the C.

[Chris22]

Hi Bob1,

 

I see what you mean about CpG methylation occuring in eukaryotes, but I think I explained it incorrectly. What I mean to say is that the results suggest that the cytosine in the GATC sequence is methylated....not necessarily a CpG methylation....but a cytosine methylation, which is common in prokaryotes I think.

 

(As Sau3AI is Dam insensitive, but sensitive to CpG methylation overlap - the gDNA is not digested with Sau3Ai so I assumed it was CpG..but it could just be a specific GATmC methylase.)

(And as DpnI only cuts when this sequence is Dam methylated, and it does cut the gDNA, it also has Dam methylation..GmATC)

 

That was my assumption but open to others...

 

I was expecting a smear of DNA and knew I would have to optimize time and concentration to get the smear between 1-10kb.

 

The transformation method I have been using is electrotransformation, have not yet tried conjugation but maybe this is a good idea....this requires lots of host strains?

[Chris22]

Hi Phage 434

 

That's an interesting idea and one I had not considered.

 

But when you say "DNA insert" do you mean amplifying the entire vector with this PCR and modified 5-me dCTP? Or do you mean the insert (as in gene of interest, e.g GFP gene) is modified like this and ligated to a desired vector with GATC sites removed. 

 

How did you remove the CATG sites in your vector? DNA synthesis or manually?

 

 

But now that you've mentioned it, couldn't your idea also be very useful for linear DNA constructs when making knockout mutants by homologous recombination? (which I had previously tried with normal PCR products and failed)

 

Thanks to you both for your responses

[phage434]

I do this in the context of a transposon insertion, which is electroporated into the cell. But it should also work for homologous recombination, and (with a bit more work) for producing circular plasmids by ligating an amplified PCR product.

[Chris22]

Hi phage 434,

 

Great, thanks.

 

Have you published this method? or would you have a protocol? (for the reagents and times etc)

 

chris