Template:SBB-Protocols Assay3

Silver Biomineralization Assay

Here we present an assay to determine whether the AG4 Silver binding peptide is present in the outer membrane of E.coli.

Incubating Colonies

1. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
2. Plate on selective antibiotics, let incubate overnight

Picking Colonies

• For each construct you will pick and later miniprep 2 colonies
• Add 4mL of LB media with the appropriate antibiotics to a clean test tube
• Pick a well-isolated, round, and "normal" looking colony with a toothpick
• Drop it in the test tube
• Incubate at 37 overnight

Treating with Silver

1. Centrifuge the colony solution for 30 seconds
2. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
3. Add 50 uL of Tris buffer

1. Wash with Tris buffer
2. Incubate in 0.1 mM silver nitrate for 24-48h at room temperature
3. Centrifuge the solution
4. Look for reddish colour change

Controls

1. Chemically reduce silver nitrate to precipitate silver (tells us silver nitrate is precipitating)
2. Lyse cells over-expressing AG4 and run assay (tells us if AG4 is precipitating silver)
3. Wash non-AG4 cells with silver nitrate and look for colour change (tells us if precipitation occurs without AG4)