Fusion Protein Want to Coat Bacteria with Proteins of Interest Surface Display: Fusions to Membrane Proteins Strept Binding Peptides Histidine Tag Random Library (Peptides) Targeted Antibodies Cellular Targeting OmpA

Surface Display: OmpA – Outer Membrane Protein A Pautsch A, Schulz GE. High-resolution structure of the OmpA membrane domain. J Mol Biol Apr 28;298(2): OmpA Outer Membrane Protein Llp Leader OmpA BioBrick Insertion Site Llp Leader 2 OmpA BioBrick Insertion Site BioBrick Insertion Site

Week 0: Transformation of 6 constructs, Plasmid Mini and MidiPreps, Restriction Digests  Week 1 – testing assays, library construction Documentation and Podcasts

Y E. coli Target Cell / Particle/ or Protein Primary Antibody Y Secondary Antibody Attached to Magnetic Particle Y E. coli Y Y Y If interactionIf no interaction

RBS Sensors RBS SensorResponse Element Assay Sensors -respond to external commands -Can be used to turn genes on and off 3. Environment Responsive Promoter 1. Cytoplasmic Regulatory Proteins 2. Two-Component Systems Triggered “Kill Switch” for safety Optimization

Week 1 Sequence Plasmids Design 3 rd Surface Expression Construct (Internal Insertion Site in Loop) Construct Library – Ligate into 2 Surface Expression Plasmids Evaluate Assay for Binding Using Control Peptides (Strep binding, His tag) Pick Targets Week 2-9 Optimize Binding Assay (Magnetic and FACS) Optimize Transformation (library coverage) Identify Peptides that Target Bacteria to Particular Cells, Viruses, or Proteins Signaling Cascade (Triggered by binding), Safety Kill Switch, Single Domain Ab Week 10 - Final Presentation and Poster Preparation