Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)

• Cumate-based gene expression system was modified for Escherichia coli.
• This system was less toxic to hosts with 2.5-fold increment in GFP expression.
• This system was more efficient in expressing GFP at low temperature.

A 474-bp ds-DNA linker-A containing all bio-parts was designed to construct tightly inducible expression vector, pNB1, for Escherichia coli. The cymR repressor expressed constitutively under pGapA promoter binds cumate operator, thus repressing the expression of target gene under T7 promoter. The potential of pNB1 was evaluated by comparing the fluorescence of green fluorescence proteins (GFP) in pNB1 and other expression system induced with cumate and isopropyl β-d-1-thiogalactopyranoside (IPTG), respectively. Compared to IPTG, cumate was found to be less toxic to bacterial cells with 2.5-fold increment in GFP expression. In addition, pNB1 was efficient in expressing GFP at low temperature.