Cellular Targeting Surface Display Fusions to Membrane Proteins

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

Surface Display: Omp. A – Outer Membrane Protein A Llp Leader Omp. A 1. Llp Leader 2 Bio. Brick Insertion Site Omp. A 2. Bio. Brick Insertion Site Pautsch A, Schulz GE. High-resolution structure of the Omp. A membrane domain. J Mol Biol. 2000 Apr 28; 298(2): 273 -82. Omp. A Outer Membrane Protein 3. Bio. Brick Insertion Site

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

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

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

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