Engineering genetic reporters for fluorescence imaging in anaerobic systems
The limited solubility of oxygen (7.6 ppm in air-saturated water) coupled with its high reactivity and restricted transport in (poorly vascularized) biological tissues can lead to the rapid emergence of anaerobic zones in various ecological and physiological niches. For instance, the mammalian gut is almost entirely anaerobic supports a complex (largely anaerobic) microbial ecosystem that profoundly impacts health and provides a unique resource for sustainable biomanufacturing. Despite the physiological, ecological, and industrial significance of hypoxia, our ability to study cells in oxygen-depleted conditions is hindered by a lack of biomolecular reporters that can function in anaerobic environments. Traditional reporters such as the green fluorescent protein (GFP) and luciferase strictly depend on oxygen to emit light. To tackle this challenge, we integrate molecular & metabolic engineering, kinetic modeling, and directed evolution to engineer bright and variously colored oxygen-independent fluorescent reporters and sensors for anaerobic imaging.
Engineering genetic reporters for magnetic resonance imaging
Genetically encoded reporters based on GFP provide one of the most sensitive and selective approaches for molecular imaging in intact cells. Unfortunately, optical techniques provide limited access to deep tissues due to poor penetration of light. In contrast to optical techniques, tissue-penetrant modalities such as magnetic resonance imaging (MRI) provide unfettered access in living animals. Unfortunately, MRI lacks the molecular precision conventionally reserved for optical reporter genes. Furthermore, relatively few MRI reporters have been turned into biochemically responsive sensors. This is a major challenge for biomedical research where animal models are routinely used to study processes such as tumor metastasis and neural function in their in vivo context. To address this challenge, we engineer biomolecules to develop sensitive and bio-responsive MRI reporters for detecting various aspects of physiological function such as tumor gene expression, metabolic activity, and neural signaling.
Support: We gratefully acknowledge generous sponsorship from the California NanoSystems Institute (CNSI), Brain & Behavior Research Foundation, Institute of Collaborative Biotechnologies (ICBT), National Institutes of Health (NIH), Department of Defense (DoD).