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bioRxiv · 10.64898/2026.04.03.716437

Surface Display For Phage Assisted Continuous Evolution: A Platform For Evolving / Screening Nanobodies In Prokaryote Systems

Flores-Mora, F. E.·Brodsky, J.·Cerna, G. M.·Tse, A.·Hoover, R. L.·Bartelle, B. B.
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Abstract

Despite >50 years of methods development, specific antibodies are still generated at low throughput and remain in high demand across biotechnology. Most biologics and immunoprobes are monoclonal antibodies, developed using a combination of inoculating animals with a target antigen, engineered candidate libraries, and multiple rounds of selection using phage or yeast display. Here we introduce a synthetic biology scheme to eliminate the need for nearly all of these steps, by combining Surface display on E. coli and Phage display with the microvirus {Phi}X174, Assisting Continuous Evolution (SurPhACE). Instead of building libraries for screening, SurPhACE runs a closed evolutionary program. A typical experiment can have 1011 mutant candidates under active selection, with complete turnover of the mutant population every 30min, or >5x1012 unique mutants per day, using less than 100mL of bacterial culture media. We demonstrate SurPhACE for optimizing a nanobody to a related epitope, and develop novel nanobodies for an arbitrary target using a minimal starting library to establish a proof of concept and identify best practices for this scalable method for generating protein binders.

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Flores-Mora, F. E., Brodsky, J., Cerna, G. M., Tse, A., Hoover, R. L., Bartelle, B. B.. 2026-04-04. Surface Display For Phage Assisted Continuous Evolution: A Platform For Evolving / Screening Nanobodies In Prokaryote Systems. https://doi.org/10.64898/2026.04.03.716437

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