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.