An amber suppressor variant of Bacillus subtilis tyrosyl-tRNA was constructed and placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter. Addition of IPTG resulted in a 50-fold increase in the expression of an rpsD-lacZ fusion containing a UAG amber codon. This system permitted isolation of a conditional lethal mutant which required IPTG for growth.

BACKGROUND For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducibl...

BACKGROUND Inducible expression systems are frequently used for the production of heterologous proteins. Achieving maximum product concentrations requires induction profiling, namely the optimization of induction time and inducer concentration. However, the respective experiments can be very laborious and time-consuming. In this work, a new approach for induction profiling is presented where in...

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-β-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 ...

In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. T...

The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 [pSH101] was characterized and compared to that of a similar culture induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a le...

BACKGROUND Heterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of t...