FAQ: What are the strain properties (C2984)?

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: (F' Δ(lacZ)M15 makes omega-fragment of β-gal; Δ(lac-proAB) 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 which 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 plus: (recA+) E. coli has a repair system that will recombine homologous sequences. Although genomic clones often have duplicated regions, they are generally less than 200 bp. The RecA repair system will not cause rearrangements or deletions under these circumstances. Strains which have the recA function intact tend to be more healthy and to grow faster than recA- strains.

Endonuclease I Deficient: (endA1) 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(hsdS-mcrB)5) Wild type E. coli K12 strains carry the EcoK Type I 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 of hsdS eliminates both the endonuclease and methyl-ltransferase activities of EcoK.

Partially Methyl Restriction Deficient: (Δ(hsdS-mcrB)5) E. coli has system of enzymes encoded by mcrA, mcrB and mrr which will cleave DNA with methylation patterns typical in eukaryotic cells. 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. This strain has functional McrA and Mrr endonucleases and may not be suitable for direct cloning of eukaryotic DNA.

M13 phage sensitive: (F´) Infection by M13 and other similar phage requires E. coli surface features conferred by the F plasmid carried by some E. coli strains. Infection by these phage allows production of single-stranded DNA and the generation of phage display libraries. The F plasmid is frequently modified to carry other useful DNA in the cell [e.g. Δ(lacZ)M15 in this cell line] and when modified is called F´.

T1 Phage Resistant: (fhuA2) 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.

Lac Promoter Control: (lacIq) The Lac repressor blocks expression from lac, tac and trc promoters frequently carried by expression plasmids. If the level of Lac repressor in E. coli cells is not sufficient to inhibit expression via these promoters during transformation or cell growth, even low levels of expression can reduce transformation efficiency and select against desired transformants. The extra molecules of Lac repressor in lacIq strains help to minimize promoter activity until IPTG is added.

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