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

the medium

Standard BioBrick plasmid backbone

Peptide secretor cassette

origin antibiotic resistance

XE PS

(http://partsregistry.org/Plasmids)

Cassette Structure

GFP

Secretory signalRBS M Peptide His

Standard Promoter

• e.g. J23100 – Strong, constitutive promoter

GFP

Secretory signalRBS M Peptide His

Standard RBS

• e. g. B0034 – star 1 and W

GFP

Secretory signalRBS M Peptide His

Synthesize Secretory Signal

• e. g. lipA or sacB – identified from registry• Possibly need to synthesize• Leave as an exercise for the student!

GFP

Secretory signalRBS M Peptide His

Some random gene

• Need a signaller…• … and a handle• Allow student contributions for alternatives

GFP

Secretory signalRBS M Peptide His

• Need:– 1 cleavage site– 1 target peptide– 1 purification handle

The ‘slot’ in the cassette

GFP

Secretory signalRBS M Peptide His

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 signalRBS M Peptide His