Optimization of Library Generation Protocols for Fluorescence-Activated Cell Sorting

• Nastassja Corrado, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware

A crucial regulator of protein homeostasis is the N-degron pathway, where a protein’s half-life is dictated by 5-10 amino acid motifs found at the N-terminus. Despite the work dedicated over the past thirty years to describing various N-end rule pathways in cells, there remains a potential to harness and discover key principles about this pathway’s machinery. Improvement of limited substrate-identifying technologies would bolster these efforts, as current methods rely on low-throughput pulldown assays that cover a small sequence space. To determine the substrate specificity of the pathway, we have developed a dual plasmid system for the discovery and screening of potent degrons in E. coli. Here, we outline our efforts to adapt this system for high-throughput degron sequence screening using fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS).

We first optimize transformation efficiencies to cover a 1E6 library size by transforming our dual reporter construct directly into a protease carrying strain by varying several parameters: assembly volume to be transformed, cell density, DNA volume in assembly, assembly inoculation time, and recovery media. Through several iterations, colony-forming units (CFUs) were near zero on all counts. We then shifted our focus to utilization of a specialized cloning strain with subsequent transformation into our desired protease deployment strain. Testing of the same parameters confirmed a set of conditions best suited for high-efficiency transformations ten-fold higher than our library size: 5 mL inoculum optical density of 0.3-0.4, a 200 μL resuspension volume following 10% glycerol washes, dialyzed Gibson assemblies, 1 μL plasmid DNA to be transformed, and SOC recovery medium. Future work would include further protocol optimization to achieve even higher transformations efficiencies. This would allow for study of larger library sizes, and thus examination of N-degron sequences of greater length.