Are relaxed plasmids origin of rep sensitive to bp substitution in RNA I/II sequ - (Feb/12/2016 )
I am working with a bacteria that has an active restriction-modification (RM) system against a commonly occurring bp sequence, GATC.
This requires me to remove the GATC sequence from the plasmid I am working with, so it will not be digested after transformation. (no commercial methylase available for GATmC)
This is not a problem for ORFs in the plasmid as they accept degenerate codon substitutions in the sequence pretty well.
However, the E.coli replicon of this vector is based on a pBR322 plasmid, which means the origin of replication is based on RNA I and RNA II primer initiation of replication (back to the old molecular textbooks for this stuff).
My question is essentially this:
If I remove/replace GATC sequences occurring within the ori (RNAI and RNAII sequence) will this completely inhibit plasmid replication in E.coli?
Is there a degeneracy in ori which allows bp substitution? If so, for my purposes I would only need to change a single base to remove the RM recognition motif, so are A/T or C/G more tolerated?
I know this is a pretty specific question but someone who knows a lot about relaxed/stringent plasmid replication might just have tried changing up the sequences.
Thank you in advance.
Plasmids replicated by most E. coli strains will be Dam methylated anyway, so your replacement might not be something necessary.... If you are not planning on replicating the plasmids in your target organism, then the native methylation might take care of your problem. You could probably go with one of the other ori sequences or replicate your plasmid in another organism.
I only have a general knowledge, so take what I say with come caution...
1)Removal/replacement probably won't affect replication as the methylation usually occurs post-replication.
2)Oris are usually A/T rich sequences, I don't know if there are degenerate sequences. but I think A/T substitutions would be more tolerated as oris are sites of melting of the DNA strands.
Bob1 - the RM (Sau3AI type) system here methylates the C not the A, and is quite distinct from the DAM methylation in coli. I have had this problem as well. Can you PCR the fragment of interest? Although the GATC methylase is not available, there is 5-methyl dCTP available, which can substitute for dCTP in a PCR reaction, directly producing methylated DNA. This could be used to transform. We could also petition NEB to supply Sau3AI methylase. One of the issues is that I believe it is lethal to coli, so you need a different organism to isolate it.
I believe you can probably do a one-base modification of the pBR322 ori to get rid of the GATC sequence without problem.
Thank you for your replies Bob1 and phage434.
I think you might be right that T/A substitution will be more tolerated.
As phage434 mentioned the typical dam methylation (GmATC) used by E.coli should not be an issue here, it is the additional cytosine methylation on this sequence (GmATmC) that, if not present on my plasmid, will be digested by an RM system.
So this is why I am choosing to remove these sequences, but I am concerned that the literature suggests Dam methylation and the GATC sequence is important in initiation of replication (oriC) sites, especially in theta replicon plasmids.
I had considered the dCTP PCR as you mentioned, but when I methylated with M.ssl (mCpG) this had no effect and I know that the bacteria actually has a typeIV RM system homolog which could unluckily recognize another cytosine methylation in another sequence. It seems specific methylation rather than general are the way to go.
I would certainly consider petitioning NEB to supply Sau3AI methylase, I had not thought of this. Have you done this before? What would we need?
No, I have not, though I have mentioned it to them in the past. We should gang up on them. The person to start with is Breton Hornblower. I'll email him now.
Apologies for the delay, will PM rather than giving our contact information online