Figure 2

Regulating target gene expression levels using the Lemo21(DE3) strain. Lemo21(DE3) is a BL21(DE3) derivative harboring the pLemo plasmid. In Lemo21(DE3), expression of the gene encoding the target protein is driven by T7 RNAP. The gene encoding T7 RNAP is located on the chromosome. Its expression is governed by the not well-titratable and very strong, IPTG inducible lac UV5 promoter. The activity of T7 RNAP can be modulated by expression of the gene encoding the natural inhibitor of the T7 RNAP, T7 lysozyme, from pLemo. The pLemo plasmid has a p15A ori and a chloramphenicol resistance marker. Expression of the gene encoding the T7 lysozyme is governed by the well-titratable rhamnose promoter. The gene encoding the target protein is located on a pET-vector. pET-vectors have a ColE1 ori and the version used in this study has a kanamycin resistance marker. The expression of the gene encoding the target protein from the pET-vector is governed by the T7lac promoter. The expression levels of the gene encoding the target protein can be increasingly dampened by the addition of increasing amounts of rhamnose to the culture. The more rhamnose is added the more T7 lysozyme is synthesized (see immuno-blot of T7 lysozyme on the right). As a consequence, T7 RNAP is increasingly inhibited and the expression levels of the target gene decrease (see inset).