Fig. 3

Biopanning experimental design schematic. Biopanning is a cyclical process that begins with subculturing the naive, unsorted library. Co-expression of cytosolic DsRed and membrane-localized peptides from the pB33-dsRed-eCPX3.0 library is achieved by induction with L-arabinose during log phase of cell growth. The induced library is then incubated with PLA 3D printing plastic for 15 min with shaking. Next, the PLA coupons were washed vigorously with PBS 1% Tween for 30 min to remove unbound cells, and then placed in LB +Cm with 2% D-Glucose media to proliferate and recover the bound library. The presence of glucose halts the production of new copies of membrane-displayed peptides, which are slowly diluted during cell proliferation, along with the cytosolic DsRed. The process is repeated for sorting rounds 1 and 2, changing only the starting library material (using the previous sorting round). After each round, NGS sequencing was performed and peptide expression level monitored. Bound cells can be visualized directly on the PLA using fluorescence microscopy due to the presence of DsRed, and binding level can also be indirectly compared using regrowth assays and cell counting