Engineering Orthogonal Luciferin:Luciferase Pairs
Monique R. Reyes
Mentor: Jennifer Prescher
Bioluminescence is an imaging technique used to non-invasively image biological processes in vivo. This
process uses an enzyme (luciferase), which catalyzes the oxidation of a small molecule substrate (luciferin)
resulting in emission of a photon of light. Bioluminescence is useful for macroscopic imaging small mammals
such as mice due to its inherent low background. Previous work has been limited to using only naturally
occurring luciferase:luciferin systems in bioluminescence imaging. Native luciferase systems are limited to
only two commonly occurring, orthogonal systems. I am working on developing new luciferase enzymes
which utilize novel luciferin analogs in order to expand the number of luciferase:luciferin pairs. A region of
the luciferase gene, coding for amino acid phenylalanine, was targeted. We found that a site directed mutation
in this region yielded emission of a photon of light, two orders of magnitude times more than the wild type
enzyme luciferase when bounded to luciferin. Semi-rational techniques were used to design new luciferase
libraries, these include a combination of random mutagenesis and site directed mutagenesis. The mutant
enzymes were evaluated through screening for improved light emission. Generating new luciferase:luciferin
pairs will broaden the utilization of bioluminescence imaging in order to improve visualization of cellular
processes in whole organisms.