Yoesep, D. J. | Search bioRxiv

bioRxiv2021

A high-throughput, automated, cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this gap by developing an automated workflow combining cell-free DNA template generation, protein synthesis, and high-throughput binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to 119 published SARS-CoV-2 neutralizing antibodies and demonstrate rapid identification of the most potent antibody candidates.

synthetic biology↗

bioRxiv2019

In vitro ribosome synthesis and evolution through ribosome display

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

synthetic biology↗