Bacillus anthracis spores are currently detected by established but slow microbiological test procedures. The development of faster, cheaper and higher-throughput detection methods would enable more effective responses to bio-terrorism and natural infectious disease outbreaks. Our goal is to engineer a reporter enzyme so that is inactive until it encounters a pathogen marker. Our design strategy is to imitate the twostep natural evolution of """"""""intrasteric"""""""" regulation. We have already generated a variant of the Escherichia coil beta-galactosidase (BGAL) that is specifically activated 5.7-fold when co-expressed with the human immuno-deficiency (HIV) protease. We believe that the E. coil alkaline phosphatase (AP) has even greater potential as a biosensor, and propose studies with the following specific aims: 1. to isolate effector-dependent AP variants with greater response to the HIV protease (>570% activation) and more robust enzyme activities. 2. to """"""""re-program"""""""" the best biosensor so that its activity becomes dependent upon the B. anthracis Lethal Factor (LF), the anti-influenza hemagglutinin (HA) antibody, or the anti-Yersinia pestis F1 antibody. 3. to array biosensors that respond to different effectors upon a chip for the rapid detection of pathogen markers. The biosensors generated in this study will streamline disease diagnosis by supplanting time-consuming and expensive immunoassays. These experiments will test the feasibility of our evolutionary hypothesis and demonstrate the utility of novel protein engineering techniques. ? ?
