When is making unmethylated DNA necessary?
Under one of three circumstances:
- To cut plasmid DNA (grown in E. coli) with a restriction endonuclease sensitive to E. coli K-12 methylation patterns (Dam [G meATC] or Dcm [C meC(A/T)GG]). Examples are BclI (NEB #R0160), (TGATCA) or ScrFI (NEB #R0110) (CCNGG). For a list of endonucleases sensitive to these, please click here.
- To transform shuttle plasmid DNA grown in E. coli into a strain of eubacteria or archaea that restricts DNA with this methylation. Genera reported to restrict methylated DNA include Streptomyces, Acholeplasma, Bacillus, Brevibacterium, Corynebacterium and Haloferax (1-6).
- To study the effects of methylation itself, (e.g. on expression or DNA repair (7-12).
What strains can be used for this?
Any otherwise suitable strain that lists dam and dcm in its genotype.
- For routine use, NEB offers ready to use competent cells, dam-/dcm- competent E. coli (NEB# C2925).
- For M13 or when LacI repression is needed, JM110 is available from (ATCC ). JM110 carries an F' that bears lacIq DEL(lacZ)M15, and is thus able to grow M13 derivatives, repress high-copy lac promoters and complement a-fragment expressed by plasmids and phagemids derived from pUC vectors.
- When recombination between repeated sequences is a problem, GM2929 is an alternative (available from the E. coli Genetic Stock Center at Yale University (CGSC Web site). This strain is ER2925 recF. Strains with recF are as defective in plasmid-by-plasmid recombination as strains with recA (9). Mutations in recA can't be combined with dam because of DNA repair problems (13, 14).
- A review of available Dam- and Dcm- strains can be found in ref. 15.
- Note that all enteric bacteria express Dam, as do cyanobacteria and Haemophilus spp refs. (16, 17) Derivatives of E. coli B, such as BL21(DE3) (NEB #C2527), T7 Express (NEB# C2566) and NEB Express (NEB# C2523), naturally lack Dcm. Most strains used by molecular biologists are derivatives of K-12, and possess both Dam and Dcm unless otherwise noted.
Are there any special considerations when working with dam-/dcm- strains?
- We do not recommend these strains as a host for primary cloning/ligation. The dam mutation can result in an increased mutation rate in the cell (18, 19). This is because Dam affects mismatch repair and the timing of the initiation of DNA replication. The dam mutation will also reduce the transformation efficiency of competent cells. DNA should be maintained in a Dam+ strain unless there is a specific need for DNA free of Dam or Dcm methylation.
- dam-/dcm- Competent E. coli (NEB #C2925) has a low but noticeable frequency of kanamycin resistant mutations. We have observed a low, variable number of spontaneous Kan resistance mutants in this strain, and also in another commercially available dam-/dcm- strains. In both strains, tiny colonies appear on Kan plates at a frequency ranging from 1 in 106 to 1 in 107 cells. These tiny colonies will not grow upon restreaking on a fresh Kan plate. This level of background resistance should not be a problem for routine transformation of plasmids carrying KanR. The reason for the strain-dependent appearance of incomplete KanR is not known. Mutations in tufG can yield resistance to low levels of kanamycin (ref. 20). There are also known interactions between ribosomal mutations leading to streptomycin resistance (rpsL, mutated in both tested strains) and tufG. Hypothetically, a defect in drug transport could reduce the effective intracellular kanamycin concentration, enabling low-level resistance mechanisms to be effective. Both tested strains carry a tsx mutation; this gene encodes a channel protein in E. coli that functions in permeation of nucleosides across the outer membrane. However, this porin is not known to play a role in aminoglycoside permeation.
Are there other tips for making unmethylated DNA?
- Good microbiological practice. Always begin with a single colony when growing the strain to make competent cells. Dam+ revertants can arise (expected revertant frequency in an overnight culture < ~106) and when they do they will have a selective advantage and will accumulate during serial mass transfer. One subculture to get the cells to exponential phase is acceptable.
- Maintaining the dam mutation: The dam mutation in (NEB #C2925) is the result of insertion by the CamR transposon Tn9. Although not essential, it may be beneficial to maintain selection for the insertion by keeping the strain on chloramphenicol (15 µg/ml). This does not mean that serial transfers are OK if you maintain selection, however.
- Spectinomycin amplification of plasmid copy number: Some plasmids (colE1, pBR322, pACYC184 and 177, but NOT pUC derivatives) can be obtained in higher yields from C2925, as from any other strain, by using an "amplification" procedure involving inhibition of protein synthesis (15). Since (NEB #C2925) is CamR, protein synthesis is not inhibited by the chloramphenicol called for in the original procedure, so use spectinomycin instead. DO NOT TRY TO GROW C2925 ON SPECTINOMYCIN. The procedure cited is based on the repression of plasmid number by the plasmid Rop protein or analogue, and on the independence of plasmid but not host replication initiation from new protein synthesis. Basically, the host chromosome stops replicating when protein synthesis is inhibited, but the plasmid keeps replicating and will continue to replicate to very high copy if the Rop protein is not present. pUC and other very high copy number plasmids are already missing Rop and will not give improved yields with this procedure.
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