Doc-mediated cell killing in Shigella flexneri using a C1/LacI controlled expression system

Abstract

In this report we describe the development of a highly stringent and dually regulated promoter system for Shigella flexneri. Dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage P1 temperature-sensitive C1 repressor that in turn was under the transcriptional control of LacI. The level of induction/repression ratios observed was up to 3700-fold in S. flexneri. The general utility of this promoter system was evaluated by demonstrating that the bacteriophage P1 post-segregational killer protein Doc mediates a bactericidal effect in S. flexneri. This represents the first report of Doc (death on curing)-mediated killing in this Gramnegative species. ß

Development of a transcriptional regulator-based bioreporter : towards a generic selection method for novel enzymes

“…The lactose operon is also activated by the global regulator CRP (cAMP receptor protein) in response to low glucose levels 132,185,186 . A major advantage of LacI is that it has not only been studied in depth, but it has also been engineered in various aspects, for example to reduce leakiness 190 , to tighten regulation 191 , to repress in presence instead of absence of IPTG 192,193 , or to alter the ligand specificity [194][195][196][197] . And most importantly for engineering purposes, high resolution protein structures with and without ligand are available.…”