Project Summary Gene/cell therapies finally come of age thanks to the fundamental advances made by innovative and improved biotechnologies. Nonetheless, many challenges particularly toxicity and undesired/uncontrolled immune response remain. This research proposal addresses these concerns and represents a critical first step toward developing novel, more effective cell/gene therapy. Toward this end, we propose a novel synthetic biology technology termed “Environment Stimuli-Induced Proximity (ESIP)” to spatiotemporally manipulate cellular functions. The new strategy can overcome the difficulty of the established chemically induced proximity (CIP) method to mediate the translation of endogenous cellular signals into tailored cellular functions with spatiotemporal precision. To demonstrate the feasibility, we propose to develop fluorescent and chemiluminescent Fe(II) and H2O2 responsive ESIP chemical inducers because Fe(II) and H2O2 are important signal molecules, which are associated with numerous biological functions and diseases, and gibberellin (GA) and abscisic acid (ABA) are established chemical inducers with low toxicity. Specifically, we will design, synthesize, test and optimize Fe2+ and H2O2-responsive fluorescent and chemiluminescent ESIP inducers (Specific Aims 1-2) and test them in cells and construct ESIP-mediated “AND” Boolean logic gates to control the conditional production of AD therapeutic proteins (Specific Aim 3). These studies will prove that these `smart' theranostic probes possess the ability to sense endogenous Fe(II) and H2O2 specifically, and the ability to trigger the release of original bioactive chemical inducers GA and ABA, which then induce downstream biological functions such as luciferase expression and the production of neuron protective BDNF proteins. The technology will ultimately be transformed into entirely new cell/gene therapies for disease treatment with significantly reduced side effects. It is expected that this powerful and general strategy can be integrated with a variety of existing synthetic biology molecular parts and tools to build new cellular genetic and signaling circuitries to generate new functions as toolbox for biomedical research and as therapeutics for various disease treatment.