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