ABSTRACT A long-standing goal in bacterial synthetic biology is the development of therapeutic and diagnostic (theragnostic) bacteria to serve as agents for delivery of personalized medicine. For example, engineered bacteria could sense a user specified stimulus or disease cue, and be programed to react through a specific controlled response. Such engineered microbes could be applied to detect and respond to disease states ranging from inflammation (e.g. inflammatory bowel disease), to infections, and metabolic disorders where they might respond through production of a small molecule metabolite, immunomodulatory agent, or antimicrobial compound. In addition to applying these engineered bacteria in loci where they naturally reside (e.g. skin, oral cavity, gut, etc.), bacteria can be engineered to even target distant tissues such as solid tumors, or other protected compartments following oral or systemic administration. To attain these lofty goals, however, the field of synthetic biology has several critical hurdles to overcome. Natural bacterial sensing systems are not necessarily designed to respond to disease cues or molecules of interest. Or, even if they are, they often are unable to respond at the relevant physiological concentrations of these molecules with acceptable signal-to-noise ratio. The present research application focuses on developing synthetic biology strategies to build sense-act biological circuits in engineered theragnostic bacteria to permit sensing of desired stimuli and responding in a controlled manner. Such generalized tools would be invaluable for applications seeking to reverse a disease status, for diagnostic purposes, or for non-medical applications. With the help of the NIGMS MIRA ESI award, I will continue and expand my research into this field with special focus on overcoming two main research challenges that currently constrain this area: Challenge 1: How can we streamline and simplify the process of developing sensing modules in engineered bacteria for a biological molecule of interest? Challenge 2: How can we enhance the sensitivity to the desired biological molecule and amplify the response to this molecule at physiological (low) concentrations? And, can we control the response to the sensed molecule so that it happens only when the target molecule reaches a threshold concentration? Such systems will be applicable as controlled release and personalized bacterial theragnostics that will act only in the presence of certain disease cues (such as inflammation, or presence of tumor or infection, etc.) but will otherwise pass safely upon administration without eliciting therapeutic action. Impact: Developing synthetic biology tools and strategies to streamline engineering of bacterial theragnostics that can respond to specific biological molecules is an important step towards a future of personalized precision medicine. These tools will also facilitate better understanding of the biosynthesis and metabolism of the target biological molecules by both host and microbes and their role in human health and diseases.

PROJECT NARRATIVE Engineering microbes as diagnostics and therapeutics is an emerging field which has the potential to revolutionize the modern medicine. The current proposal is seeking to develop synthetic biology tools and strategies to advance this field and facilitate its widespread application.