T7 RNA Polymerase (lacZ::T7 gene1) T7-Express has the T7 RNA polymerase gene inserted into the lac operon on the E. coli chromosome and is expressed under the control of the lac promoter. This configuration provides controlled induction of the polymerase and consequently, inducible control of transcription of genes downstream of the T7 promoter. This system provides potential advantages over strains such as BL21(DE3), that carry the T7 RNA polymerase on a lysogenic prophage. Although λDE3 is normally dormant in the host chromosome, the induction of the SOS cascade can occur as the result of expressing proteins that damage the E. coli chromosome, either directly or indirectly. This may lead to cell lysis.
Protease Deficient ([lon] ompT): E. coli B strains are ""naturally"" deficient in the lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes. Mutations of other genes can help to ameliorate the sometimes-deleterious effects of these protease defects (e.g. sulA, below).
Recovery from DNA Damage (sulA11): E. coli cells can tolerate a substantial amount of chronic DNA damage as long as repair is allowed to proceed. This capacity is compromised if the cells are unable to divide following repair. In lon- cells, SulA, a cell division inhibitor, accumulates and causes cells to become hypersensitive to DNA damage. The sulA mutation introduced into the T7 Express strain allows cells to divide more normally in the absence of Lon protease.
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(mcrC-mrr)114::IS10) Wild type E. coli B strains carry a Type I restriction endonuclease which cleaves DNA with the site TGA(N8)TGCT. 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: (Δ(mcrC-mrr)114::IS10 and R(mcr-73::miniTn10--TetS)2): E. coli has a system of enzymes encoded by mcrA, mcrBC and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. All three Mcr enzymes and Mrr have been inactivated in T7 Express allowing the introduction of eukaryotic DNA of genomic origin (e.g. primary libraries) if desired.
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.
LacIq strains also contain:
Lac Promoter Control (lacIq): The Lac repressor blocks expression from lac, tac, trc and T7-lac promoters frequently carried by expression plasmids. If the level of Lac repressor in E. coli cells in not sufficient to inhibit expression via these promoters during transformation or cell growth, even low levels of expression of toxic genes 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.
LysY strains also contain:
T7 Lysozyme (lysY): This strain expresses T7 lysozyme variant K128Y which lacks amidase activity, yet retains the ability to inhibit T7 RNA polymerase. Basal expression of the target gene is minimized without inhibiting IPTG-induced expression. The lysY gene is carried on a single-copy miniF plasmid.