Date of Completion

Spring 5-1-2015

Project Advisor(s)

Paulo Verardi; Daniel Gage; Guillermo Risatti

University Scholar Major

Pathobiology

Abstract

Vaccinia virus (VACV) is well known for its use as the vaccine in the successful campaign to eradicate smallpox and a powerful vector for vaccines, immunotherapies, and oncolytic viral therapies. Advancements in synthetic biology have recently led to the development of synthetic gene circuits, which can use recombinases to respond to inputs with logic and memory. We propose that this technology can be employed to make “logical” VACV vectors which could be programmed to change their actions based on sensory inputs for use in the development of safer vaccines or oncolytic viral therapy agents which selectively lyse cancer cells. In this project we tested the functionality of recombinases Bxb1 and PhiC31 in synthetic VACV circuitry. We developed simple synthetic circuits with VACV promoters wherein each recombinase can be induced under lac or tet operon elements to irreversibly invert a promoter, which switches fluorescent reporter expression from red to green and can be observed in VACV infection/transfection assays. We detected only red fluorescence with little to no green in cells transfected with bxb1 plasmids, suggesting that no promoter inversion occurred and Bxb1 may not be functional in VACV-infected cells. Green fluorescence indicative of promoter inversion was detected in all cells transfected with phiC31 plasmids. This occurred regardless of absence of inducer, suggesting PhiC31 is highly functional in VACV-infected cells but cannot be regulated by TetR or LacI repressor proteins, even when they are pre-expressed at high levels. Future recombinase-based synthetic circuitry in VACV will thus require tighter repressor systems.

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