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.
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.