Date of Award

10-3-2023

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

First Advisor

Edward Campbell

Abstract

Bioluminescent imaging systems (BLI) have become a valuable tool for doing real-time in vivo studies. BLI systems typically allow for the imaging of surface and subsurface level tissues but as depth increases, the resolution decreases while the signal to noise ratio increases. Deep tissue imaging is therefore a major limitation for BLI. To address this limitation there has been a consistent movement in luciferase research to create long wavelength emitting luciferases that still have a high photon emission rate. Longer wavelengths of light are able to penetrate deeper tissues due to having a lower amount of absorbance and scattering from the tissues. In recent papers, Zambito et al. 2021 and Hall et al. 2018, they had described a modified click beetle luciferase (CBR2) and napthyl-based luciferin that when put together were able to emit at 730nm. They also showed that by using an unmixing algorithm, they were able to differentiate between CBR2 and a firefly luciferase signal within an in vivo mouse model. This thesis project aimed to utilize the CBR2 in a dual imaging system with a caspase-1 biosensor(C7) to track the progression of a bacterial infection and the subsequent inflammation. Our preliminary testing of CBR2 showed that it would not be viable for a dual imaging system. We switched tracks to mimicking the C7 biosensor, since that would allow for the ability to image deep tissues for inflammation such as within the brain. We replicated the circular permutation present in the construction of the C7 biosensor in CBR2. We also inserted a caspase-1 cleavage sequence, IQAD, in the linker sequence connecting the N and C fragments. Our tests showed that the new construct, pCBR2, was only partially functional. It showed an inability to sense inflammation and was enzymatically functional whether the stimuli for inflammation were present or not. We then tried multiple variants of pCBR2 to solve this issue, but we were ultimately unsuccessful. However, these studies establish a foundation for future research into the utility CBR2 as an inflammation biosensor.

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