title: Stabilizing proteins through saturation suppressor mutagenesis
design: CcdB second-site saturation suppressor mutagenesis library is displayed on the yeast cell surface. The library is sorting in different bins based on expression and binding to its ligand and MFI for each mutant is reconstructed.  Yeast cells were grown overnight in SDCAA media and the culture was diluted to a final OD(600) in a fresh SDCAA media. The cells were grown to a final OD of 2, and induced in SGCAA media for 16 hours. Sorted cells were grown on SDCAA agar media and the colonies were scrapped. Pooled plasmids were purified and the gene of interest of was PCR amplified using bins specific barcode. Equal amount  of PCR products from different bins were pooled for sequencing.  Sequencing library was generated using TruSeq DNA Sample Sample Preparation Guide, Part # 15026486 Rev. C.