Orthogonal split luciferases for imaging multiplexed cellular behaviors
Ended Project Grant Awarded Last action
- Award ID
- R15GM151711
- Recipient
-
Dickinson College UEI G4KQDKZBL848
- Awarding agency
-
National Institute of General Medical Sciences
National Institutes of Health, Department of Health and Human Services
- Obligated
- $413,870
Award facts
- Assistance listing
- 93.859 Biomedical Research and Research Training
- Completion date
- Place of performance
- Claremont, CA 91711, USA
Award details come from USAspending.gov.
Description
- Abstract
- Disease states are characterized by complex interactions that occur between a variety of cell types and biomolecules in an organism. Researchers have turned to bioluminescence imaging (BLI) to track the behavior of these cells and understand the underpinnings of disease and the development of effective treatments. BLI utilizes the light-emitting ability of bioluminescent enzymes to sensitively illuminate individual cells without the need for surgery. Because organisms do not glow, BLI is exquisitely sensitive, with the ability to detect as few as one glowing cell in the body of a mouse. This has enabled researchers to study the efcacy of cancer-killing drugs, the location and progression of infection, and the success of stem cell treatments. Despite its ubiquity in the ELD, BLI of multiple different cell types simultaneously remains difcult. Our research seeks to further expand the utility of this tool through a unique approach to multicomponent bioluminescence imaging. To accomplish this, we will repurpose a split bioluminescent protein called nanobit. Nanobit comprises a heterodimer binding pair made up of a small peptide, called smbit, and a larger protein subunit, called lgbit. Nanobit is only capable of light emission when smbit and lgbit bind. In aim 1 of the proposal, we will use protein engineering and directed evolution techniques to produce orthogonal smbit-lgbit binding pairs. Libraries of lgbit enzymes will be cloned, and a panel of smbit peptides will be synthesized. High-throughput techniques will evaluate the light emission of each lgbit mutant in combination with each smbit peptide. "winning" mutants will be sequenced via next generation sequencing (NGS) and mutations will be combined to form optimized orthogonal probes. In aim 2 we will test our new orthogonal nanobit probes in mammalian cells, tissue models, and in the bodies of live mice. First, to improve the tissue penetration of nanobit light emission, we will modulate the color of bioluminescence by appending small molecule uorophores to our smbit peptides. Stable mammalian cell lines containing our probes will next be tested with our smbit-uorophore probes in tissue models and in live mice. Probes will be judged by their sensitivity and selectivity. This work will represent the RST effort to adapt nanobit for multicomponent imaging. Our protein engineering data will be immediately useful to the bioluminescence imaging community. Further, these probes will be useful for imaging protein-protein, host-pathogen, and cancer-immune cell interactions.
Transactions
| Action | Obligated | Date |
|---|---|---|
| Amendment | $0 | |
| Amendment | $379,558 | |
| Amendment | $379,558 | |
| Amendment | -$348,270 | |
| Amendment | -$348,270 | |
| Original award | $351,294 |
Quick answers
Who received award R15GM151711?
R15GM151711 was awarded to Dickinson College by National Institute of General Medical Sciences (National Institutes of Health) on September 26, 2023.
How much is award R15GM151711 worth?
$413,870 has been obligated on R15GM151711.
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