FAQ: What are the strain properties (C3020)?

The properties of this strain that contribute to its usefulness as a cloning strain are described below. The genotypes underlying these properties appear in parentheses.

Blue/White Screening: (Φ80 Δ(lacZ)M15 makes omega-fragment of β-gal; lacX74 deletes the β-gal gene on the chromosome) pUC19 and similar plasmids code for the α-peptide of β-galactosidase (lacZ). The α-peptide can combine with the omega-fragment of β-galactosidase that is carried on the F' (α-complementation). When β-galactosidase is reconstituted in this manner it can cleave X-gal and results in blue colonies on an X-gal plate. Inserts cloned into the plasmid polylinker disrupt the α-peptide gene and the colonies are white.

Recombination Deficient: (recA) E. coli has a repair system which will recombine homologous sequences. Genomic clones often have duplicated regions, and RecA mediated rearrangements can be problematic, particularly when regions of homology are longer than 50 bp. Strains that are recA- tend to grow more slowly than recA+ strains.

Endonuclease I Deficient: (endA) The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations.

Restriction Deficient: Δ(mrr-hsdRMS-mcrBC) Wild type E. coli K12 strains carry a restriction endonuclease which cleaves DNA with sites (AAC(N6)GTGC and GCAC(N6)GTT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The deletion described above eliminates both the methylase and the endonuclease.

Methyl Restriction Deficient: mcrA Δ(mrr-hsdRMS-mcrBC) E.coli has a system of enzymes, mcrA, mcrB and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. DNA derived from PCR fragments, cDNA or DNA previously propagated in E.coli will not be methylated at these sites and will not be cleaved. All three Mcr enzymes have been inactivated in NEB 10-beta allowing the introduction of eukaryotic DNA of genomic origin (e.g. primary libraries) if desired.

T1 Phage Resistant: (fhuA) T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell.