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Fig. 1 | Microbial Cell Factories

Fig. 1

From: The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli

Fig. 1

Construction of pReX. a The Lemo21(DE3) setup is based on two plasmids; a T7-based pET-expression vector and pLemo. The T7-based pET-expression vector, which has a T7 promoter that contains a lac-operator site (T7lac), a kanamycin resistance marker (Km-R) and a pMB1 origin of replication, is used for the expression of the gene encoding the target protein (gene of interest). pLemo is used for the l-rhamnose inducible PrhaBAD promoter-based expression of the gene encoding the T7 lysozyme (T7lys). In addition, pLemo contains the rhaSR genes encoding the regulatory proteins RhaS and RhaR, a p15A origin of replication and a chloramphenicol (CmR) resistance marker. b pReX was created by incorporating the part of pLemo containing the gene encoding the T7 lysozyme along with the l-rhamnose inducible PrhaBAD promoter governing its expression and the rhaSR genes in a pET-vector with a kanamycin resistance marker (KmR) (details of the construction of pReX can be found in the “Methods” section). The idea behind pReX is that it can be used to modulate the activity of the T7 RNA Polymerase (T7 RNAP) in BL21(DE3) and derivatives thereof by adding different amounts of l-rhamnose to the culture medium, which results in the controlled production of the T7 RNAP inhibitor T7 Lysozyme (T7LysY). Notably, the IPTG inducible promoter (PlacUV5) governing the expression of the gene encoding the T7 RNAP in BL21(DE3) is only poorly titratable. c pReX was transformed into E. coli BL21(DE3), and the expression of the gene encoding T7 lysozyme was induced for 4 h with varying l-rhamnose concentrations (0, 500, 1000, 2500 and 5000 µM). Subsequently, T7 lysozyme accumulation levels in whole cell lysates representing equal amounts of cells were monitored by immuno-blotting

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