Ph.D.
Candidate, Nugen Lab (https://cals.cornell.edu/sam-r-nugen)
Department
of Food Science, Cornell
University
Engineering
Recoded Bacteriophage T7 Genome for Incorporating Non-Canonical Amino Acids
& Bacterial Detection
Abstract
Synthetic biology
has made leaps and bounds in recent years, however, complete de-novo
assembly of increasingly complex genomes with multiple intentional
mutations has remained challenging and time-consuming. Golden gate assembly
is a method often used in synthetic biology to generate libraries of
diverse gene circuits, since the order of fragment assembly is easily
computed and the ease at which these can be produced in a single one-pot
reaction. Complete assembly of bacteriophage genomes is an excellent vehicle
for proving the dynamism of this technique while looking to meet the
challenges of detecting dangerous pathogens in drinking water.
Bacteriophages use highly specific targeting mechanisms for their host with
well-defined and small genomes, making them susceptible to genetic
engineering. Here we engineered a bacteriophage biosensor with a completely
recoded genome, allowing for the capsid-specific in-vivo incorporation
of a non-canonical amino acid (l-HPG) bearing an alkyne group, for
bioorthogonal cycloaddition with azide-conjugated magnetic nanoparticles.
This recoded genome will prove a useful system for the incorporation of
other ncAA and exploration of bioorthogonal processes in bacteriophage
systems. The functionalized magnetic phage particles enable highly
efficient detection of E. coli through a nanoluciferase assay using
a simple camera and lightbox apparatus, dramatically reducing the time and
cost for such assays in a field setting.