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Fusion Protein

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

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Fusion Protein

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

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

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

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

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

  6. Week 1 Sequence Plasmids Design 3rd 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

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