Bacterial overexpression of mutant 6S RNA produces growth defects and correlates with in vitro release defects. (A) Growth curves for E. coli BL21 (DE3) cells transformed with one of the following plasmid vectors; pEcoli-LowBinder, pEcoli-T1, or pEcoli-R9-33. Cells were grown in LB+Amp (100 μg/mL) and 5 mM IPTG (+ symbols) from the time of inoculation. Cells transformed with pEcoli-R9-33 but grown in the absence of IPTG (− symbols) were used as a control together with untransformed BL21 cells grown in LB broth. (B) Expression of mutant RNA correlates to cell survival and growth. Exponentially growing cells (OD600 = 0.7) in LB+Amp were plated onto LB+Amp agar with and without 5 mM IPTG. The percentage of CFU was calculated for each construct and expressed as a percentage of survival normalized to that of the pEcoli-Empty. The control plasmid pEcoli-Empty grew like pEcoli-R9-33 – IPTG in LB+Amp liquid culture (data not shown in panel A). Error bars correspond to standard deviations from the averages of three independent experiments.
