Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.
We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).
Doherty
J P
JP
Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Lindeman
R
R
Trent
R J
RJ
Graham
M W
MW
Woodcock
D M
DM
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D015698:Genomic Library; D005838:Genotype; D006801:Humans; D016296:Mutagenesis; D011995:Recombination, Genetic