Microcapillary Array-Based High Throughput Screening for Protein Biomanufacturability

Gene expression is a complex phenomenon involving numerous
interlinked
variables, and studying these variables to control expression is essential
in bioengineering and biomanufacturing. While cloning techniques for
achieving plasmid libraries that cover large design spaces exist,
multiplex techniques offering cell culture screening at similar scales
are still lacking. We introduced a microcapillary array-based platform
aimed at high-throughput, multiplex screening of miniature cell cultures
through fluorescent reporters. The clone recovery mechanism provides
100× enrichment ratios compared to traditional techniques for
establishing phenotype-to-genotype linkages. We conducted experiments
to delineate the effects of three key plasmid design featurespromoters,
5′ untranslated regions, and amino acid sequenceson
protein titer. We identified a small set of promoters that maximize
protein titer from thousands of promoters with widely varying transcription
rates. We established that mRNA half-lives, controlled by 5′
untranslated regions, correlate with protein expression. Using dual-reporter
imaging, we demonstrate relative analyses of multiple ribosome binding
sites in operons. Lastly, we discuss the effect of structural protein
hydrophobicity scores on their expression and cell growth profiles.
Through multiple experiments with libraries of plasmid constructs,
we demonstrate population binning, dual-reporter operon screening,
chemical perturbation, and cell growth estimation using brightfield
absorbance measurements with the platform.