Disulfide bond formation in the cytoplasm: Normally reductases in the E. coli cytoplasm keep cysteines in their reduced form, thereby reducing any disulfide bond that may form in this compartment. SHuffle has deletions of the genes for glutaredoxin reductase and thioredoxin reductase (Δgor ΔtrxB), which allows disulfide bonds to form in the cytoplasm. This combination of mutations is normally lethal, but the lethality is suppressed by a mutation in the peroxiredoxin enzyme (ahpC*). In addition, SHuffle expresses a version of the periplasmic disulfide bond isomerase DsbC which lacks its signal sequence, retaining it in the cytoplasm. This enzyme has been shown to act on proteins with multiple disulfide bonds, to correct mis-oxidized bonds and promote proper folding. The gene for the cytoplasmic DsbC is present on the chromosome.
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.
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).
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.