Fusion Protein

Want to Coat Bacteria withProteins of Interest

Surface Display: Fusions to Membrane Proteins

Strept Binding PeptidesHistidine TagRandom 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. 2000 Apr 28;298(2):273-82.

OmpA Outer Membrane Protein

LlpLeader OmpA

BioBrick Insertion Site

LlpLeader 2 OmpA

BioBrick Insertion Site

BioBrick Insertion Site

1.

2.

3.

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

Documentation and Podcasts

YE. coli

Target Cell / Particle/ or Protein

Primary Antibody

Y Secondary AntibodyAttached to Magnetic Particle

YE. coli YE. coli Y

Y

If interaction If no interaction

RBS

Sensors

RBS Sensor Response ElementAssay

Sensors-respond to external commands

-Can be used to turn genes on and off

3. Environment Responsive Promoter

1. Cytoplasmic Regulatory Proteins2. Two-Component Systems Triggered “Kill Switch” for safety

Optimization

Week 1Sequence 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 2-9Optimize 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