Development of Methods and Tools for Cell-Free Synthetic Biology Applications

Abstract

A primary advantage of bead surface display is the potential for highly controlled, multivalent display of immobilized protein. To realize this potential, we built a bead surface display platform with multivalency in mind. Starting with a display particle with dense functional groups, we systematically designed and synthesized a bead saturated in DNA and protein attachment sites utilizing chemoselective coupling mechanisms. With the potential of tens of millions of biomolecule attachment sites, we optimized the biological steps of genotype and phenotype immobilization empirically. Starting with a single gene copy per bead to mimic monoclonality, we amplified genotype onto the bead surface to a sufficient degree to express attachable protein and, yet sparse to leave most of the bead surface area exposed for protein attachment. We systematically rescued the in vitro transcription step to maximize protein expression from the immobilized context. Our methodology enables more insensitive and indirect detection mechanisms for rank-sort screening of protein libraries.