Simple methods for making EcoK and McrA restrictionless mutants.

Restriction systems present a major obstacle to the cloning and expression of heterologous DNAs in bacteria such as E. coli. Many modem cloning strains therefore carry disabling mutations in genes encoding restriction endonucleases to prevent degradation of these heterologous DNAs, however many laboratory strains of E. coli are not deficient in host restriction activity and are therefore inaccessible to foreign DNAs. Here we describe simple methods for genetically eliminating EcoK and McrA restriction from E. coli that are generally applicable to strain improvement programs with other bacterial species. EcoK restriction defective hsd mutants of E. coli can be produced by UV irradiation as part of the cell's SOS response and selectively enriched by transformation of the irradiated cells with plasmid which has not been modified by the EcoK system. Log phase cells are resuspended in 10 mM MgSO4 and a 2 ml sample irradiated with 30 to 60 J/m2 UV in a 5 cm plastic petri dish. The irradiated cells are resuspended in an equal volume of Luria broth and grown overnight to ensure sufficient time for fixation and expression of any restrictionless mutants. This mutagenized culture is then selectively enriched for restrictionless mutants by transformation with pBR322 DNA lacking EcoK modification using standard transformation protocols (1). The resulting transformants are screened for EcoK restriction activity by cross streaking against non-modified phage such as X. Restriction defective mutants are identified by their sensitivity to killing by a cross streak of non-modified phage. This method produces approximately 20% restrictionless mutants after 30 J/m2 UV and 40% restrictionless mutants after 60 J/m2 UV. Two thirds of the restrictionless mutants produced by this method are also modification defective as might be expected from the operon structure and activity of the products of the hsdRMS genes (2). Non-modified phage and plasmid used in this procedure are simply made by propagation on a non-modifying host such as E. coli HB101. The SOS response is also responsible for the permanent loss of McrA DNA restriction through a mechanism resembling abortive phage induction whereby mcrA genes are lost with the loss of the defective element e14 (3, 4). The procedure for inducing this loss involves the same UV irradiation protocol as outlined above. The following day the liquid culture grown from the inoculum of irradiated cells is streaked out on agar plates for growth of single colonies. The McrA restricting ability of such individual clones is determined by increased transformation ability with plasmid DNA which was methylated in vitro with Hpal methylase (3, 5). In the dose ranges recommended here approximately half of the progeny of the irradiated culture are expected to be McrA defective so that only a small number of clones need be tested to obtain a McrA defective stock. Since both of these processes for eliminating DNA restriction result from induction of the SOS response a functional recA locus is needed. This could be either chromosomal or temporarily provided by a recA plasmid such as pDR1453. Again the widespread occurrence of SOS-like responses in many species of bacteria would make these strategies generally applicable.