I have plasmid DNA (BFP in pET vector) that minipreped from DH10B and DH5apha. There is no colony when I transformed DNA (from the DH10B) to MG1655, and many colonies when I transformed DNA (from the DH10alpha) to MG1655. One of my committees suggested the restriction modification system might be the issue. Although my issue has resolved, but the mystery hasn't. I wonder if anyone can help clarify this?
Your committee member is correct. DH5 is R-M+ so it does not restrict incoming DNA but it does modify DNA and can be transformed into any E. coli strain. However DH10B is deleted for the entire hsd operon so it neither restricts nor modifies (R-M-). Therefore plasmid DNA isolated from DH10B can only be transformed into a strain that is R-. So you could transform that DNA into DH5 and then make another mini prep. MG1655 is of course wild type at the hsd locus so is R+M+. Alternately you choose another strain in lieu of MG1655.
Thank you, Michael. I just found out that my plasmid has the recognition site for the enzyme, enchoing what you said.
A follow-up question. I know some restriction enzymes fail to cut the methylated DNA. Is this the ONLY why we use DH10B? I mean DH5alpha can basically do everything what DH5alpha does.
I just want to be cautious about what cloning strain I should use in future.
There are a few restriction enzymes which are blocked by methylated DNA. Of the two E. coli systems that methylate DNA, dam and dcm, DH5a has both and DH10b is mutant for dcm but still has the dam.
However there are also systems in E. coli that that inhibit the uptake of DNA methylated at certain sites (McrBC systems). For labs that routinely do cloning from organisms whose DNA has certain methylation patterns, that might also be a reason, for example DH10b was originally developed for labs working on Staphylococcus for that reason.
Kok Zhi Lee ,
Did you use the same batch of competent MG1655 cells in both experiments and/or included an identical positive control of methylated plasmid DNA in both to control for differences in transformation efficiency?
The reason I ask is that in my experience, mismatched dam/dcm/mcr systems do decrease transformation frequency but seldom to the point of not getting any colonies.
Alejandro Martin, DH5a are R-M+ and DH10b are R-M-, so dam/dcm/mcr aside the main issue here is the hsd restriction system. But I agree you should be able to get some colonies with MG1655 but transformation will be down hundreds to thousands fold.
Alejandro,
I did use the same batch of competent MG1655 cells in both experiments. I also used another pET plasmid to do the both experiments and gave me the same results.
Thank you for asking. I did not have this issue in the past and just had this issue recently. My advisor told me that his PhD lab only uses DH10B as their cloning strain and they don't have this issue. My working hypothesis is that the mismatched dam/dcm/mcr systems-mediated issue gradually becomes dominant over time. My freezer stock was made in 2016. I even sequenced the entire plasmid and it looked fine.
Any thoughts?
Michael J. Benedik , yes you are right, sorry for the brain fart. Still, if a mismatched hsd system causes a 10E3 reduction in transformation frequency, it still does not explain the failure to get any colonies when transforming MG1655 with a plasmid, unless transformation efficiency was extremely low to begin with.
Kok Zhi Lee , when you say you used another pET plasmid and got the same results, you meant another pET plasmid from DH10B, or from DH5? Also, by "the same results" you mean it failed to transform MG1655 when purified from DH10B but not from DH5?
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