A NEW APPROACH FOR CONTAINMENT OF MICROORGANISMS - DUAL CONTROL OF STREPTAVIDIN EXPRESSION BY ANTISENSE RNA AND THE T7 TRANSCRIPTION SYSTEM

The use of microorganisms in the open environment would be of less con cern if they were endowed with programmed self-destruction mechanisms, Here, we propose a new genetic design to increase the effectiveness o f cell suicide systems. It ensures very tight control of the derepress ion of cell death by the combination of the bacteriophage T7 RNA polym erase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor, Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bact eria, based on the conditional expression of the lethal Streptomyces a vidinii streptavidin gene, Toxicity of streptavidin is derived from it s exceptionally high binding affinity for an essential prosthetic grou p, D-biotin, The entire construct was designed to allow the soil bacte rium Pseudomonas putida to survive only in the presence of aromatic hy drocarbons and their derivatives which it can degrade, Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation, The general requirement f or biotin through the living world should make streptavidin-based cond itional lethal designs applicable to a broad range of containment stra tegies.