1. Goal: To persuade a bacterium to produce a variety of small peptides and secrete them into the medium

2. Standard BioBrick plasmid backbone Peptide secretor cassette origin antibiotic resistance X E PS (http://partsregistry.org/Plasmids)

3. Cassette Structure GFP Secretory signal RBS M Peptide His

4. Standard Promoter e.g. J23100 – Strong, constitutive promoter GFP Secretory signal RBS M Peptide His

5. Standard RBS e. g. B0034 – star 1 and W GFP Secretory signal RBS M Peptide His

6. Synthesize Secretory Signal e. g. lipA or sacB – identified from registry Possibly need to synthesize Leave as an exercise for the student! GFP Secretory signal RBS M Peptide His

7. Some random gene Need a signaller… … and a handle Allow student contributions for alternatives GFP Secretory signal RBS M Peptide His

8. Need: – 1 cleavage site – 1 target peptide – 1 purification handle The ‘slot’ in the cassette GFP Secretory signal RBS M Peptide His

9. What goes in the player? The polymer engineer is thinking about, e.g. extracellular matrix proto-molecules and self- assembling elements The biologist is thinking about using this as a recruiting tool… GFP Secretory signal RBS M Peptide His