Figure 1. Isolation of Mt56(DE3) from BL21(DE3).

The exceptionally toxic target membrane protein YidC, which in contrast to other targets, does upon T7-based production not seem to lead to the isolation of mutations in P_lacUV5 due to recombination with P_lacWT of the lac-operon was used to screen for IPTG-resistant BL21(DE3)-derivatives. YidC was C-terminally fused to GFP to facilitate monitoring its production in the cytoplasmic membrane. (a) GFP C-terminally fused to a membrane protein only folds properly and becomes fluorescent if the membrane protein-GFP fusion is inserted in the cytoplasmic membrane³³. If the membrane protein-GFP fusion aggregates in the cytoplasm, the GFP moiety does not fold properly and does not fluoresce. A membrane protein-GFP fusion with a properly folded GFP moiety runs faster in SDS-PAGE than a membrane protein-GFP fusion with a not properly folded GFP moiety¹⁹,²⁶. (b) BL21(DE3) was transformed with a pET-derived yidC-gfp expression vector and after recovery of the transformation mixture at 37 °C it was plated on LB-agar plates containing the inducer IPTG (0.4 mM). Plates were incubated at 37 °C. The screen yielded quite a number of IPTG resistant colonies. The use of a T7-based yidC-gfp expression vector will not result in IPTG-resistant BL21(DE3)-derivatives with a weakened lacUV5 promoter governing the expression of t7rnap¹⁷. The only IPTG resistant colony that was fluorescent was the 56^th colony monitored for fluorescence. Therefore, the strain derived from this colony was denoted Mutant56(DE3), hereafter referred to as Mt56(DE3). (c) Cured Mt56(DE3) was retransformed with the yidC-gfp expression vector used for its isolation and, using its ancestor BL21(DE3) transformed with the yidC-gfp expression vector as a control, a plate spot test was used to check for the ability to grow in the presence of IPTG (top panel) and fluorescence (bottom panel). Fluorescence is an indication for YidC-GFP production in the cytoplasmic membrane). The dilutions spotted are indicated on top of the panels.