Recombineering Lambda Phage to Execute Futile Cycles

Abstract

Increasing bacterial susceptibility to killing by oxidants could be a useful strategy to help eliminate harmful bacteria. Previously, our lab has shown that activation of futile cycles in E. coli increases bacterial susceptibility to killing by oxidants (unpublished data). The activation of futile cycles, reactions that only result in loss of energy, is a potential strategy to combat bacterial pathogens. Currently, we have only studied futile cycles in vitro using plasmids to express proteins required for their activation, however future attempts to study their effects in vivo have to be capable of delivering the futile cycle-activating protein to the right bacteria within an infected animal. Bacteriophage could possibly be used as a system for delivery of these futile cycle proteins, since their genomes can be expressed in their bacterial hosts and would not target animal cells. Engineering the bacteriophage lambda (λ) to express a protein that engages a one-step futile cycle also allows for highly selective targeting of bacteria, which is essential to study the effects of futile cycles in vivo. Here, we use the traditional Datsenko-Wanner method of one-step gene disruption as a means to genetically engineer λ.