Mukherjee Lab

University of California, Santa Barbara

Elings Hall 3434

Santa Barbara, CA 93106-5080

 

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Engineering genetic reporters for fluorescence imaging in anaerobic systems

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 as well as provides a unique resource for sustainable biomanufacturing. Several pathogens exploit hypoxia to resist the action of antibiotics. 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. Consequently, anaerobic biosystems have remained largely “invisible” to the prevalent biomolecular imaging toolkit. To tackle this challenge, we pursue a variety of approaches integrating molecular & metabolic engineering, kinetic modeling, and directed evolution to engineer bright and variously colored oxygen-independent fluorescent reporters and sensors for anaerobic imaging.

Photosensory proteins (known as LOV receptors) emit oxygen-independent fluorescence unlike GFP

Directed evolution of LOV reporters to increase brightness

Mining microbial genomes for bright, photostable reporters

Engineering genetic reporters for magnetic resonance imaging 

 

Genetically encoded reporters based on GFP and luciferase provide one of the most sensitive and selective approaches for molecular imaging in intact cells. Unfortunately, optical techniques provide limited access to deep-seated tissues due to poor penetration of light. As a result, optical reporter genes provide limited capabilities for noninvasively studying biological function in the context of intact opaque animals. 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 for functional interrogation in animals. 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 important in vivo context. To address this challenge, we engineer biomolecules and whole cells 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.

Paramagnetic proteins generate MRI signals via relaxation enhancement of water molecules

Water transport proteins generate MRI signals by increasing diffusion of water molecules in tissues

Collaborators: Irene Chen (UCLA), Tod Kippin (UCSB), Karen Szumlinski (UCSB), Michelle O'Malley (UCSB), S. Gene Kim (NYU), Dmitry Novikov (NYU), Els Fieremans (NYU), Manish Kumar (Penn State), Isaac Cann (UIUC), Darci Trader (Purdue University), Nanyin Zhang (Penn State)

Support acknowledgements: California NanoSystems Institute (CNSI), Brain & Behavior Research Foundation, Institute of Collaborative Biotechnologies (ICBT), National Institutes of Health (NIH)