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.

Procedure

Experimental Group

JCA: Is AgNO3 not AgNO2

pBca9145-Bca1363 as control plasmid. pBca9145CA- part

test whether AgNO3 reacts with LB test whether AgNO3 reacts with TBS

Group Samples

1. 2x cell dilution. 0.1mM AgNO3. with arabinose.
2. 2x cell dilution. 0.1mM AgNO3. without arabinose.
3. 2x cell dilution. 0.05mM AgNO3. with arabinose.
4. 2x cell dilution. 0.05mM AgNO3. without arabinose.
5. 5x cell dilution. 0.1mM AgNO3. with arabinose.
6. 5x cell dilution. 0.1mM AgNO3. without arabinose.
7. 5x cell dilution. 0.05mM AgNO3. with arabinose.
8. 5x cell dilution. 0.05mM AgNO3. without arabinose.

Picking and Incubating Colonies

1. Add 4mL of LB media with the appropriate antibiotics to a clean test tube
2. Pick a well-isolated, round, and "normal" looking colony of our proposed silver binding peptide with a pipet tip
3. Drop it in the test tube
4. Incubate at 37C overnight

Cleaning

1. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
3. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
4. Add 200uL of TBS (pH 7.4) and resuspend
5. Centrifuge the colony solution for 30 seconds
6. Pour out the supernatant
7. Add 200uL of TBS and resuspend
8. Dilute the solution 10X. Measure OD of solution using spectrophotometer. (at 600nm)

Treating with Silver

1. Make 10mM stock of AgNO3.
2. Add 200uL of 0.1 mM silver nitrate and resuspend the cells
3. Incubate overnight (24-48 hours) at room temperature
4. observe color change and precipitation of colored compound
5. Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)

Control Group - non-AG4 cells

*note: do controls for each of the sample groups

Picking and Incubating Colonies

• Add 4mL of LB media with the appropriate antibiotics to a clean test tube
• Pick a well-isolated, round, and "normal" looking colony of control E coli with no silver binding peptide with a toothpick
• Drop it in the test tube
• Incubate at 37 overnight

Cleaning

1. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL)
3. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
4. Add 1 mL of TBS (pH 7.4) and resuspend
5. Centrifuge the colony solution for 30 seconds
6. Pour out the supernatant
7. Add 200uL of TBS and resuspend
8. Dilute an aliquot of the solution 10X. Measure OD of solution using spectrophotometer (at 600nm)

Treating with Silver

1. Make 10mM stock of AgNO3
2. Add 200uL of 0.1 mM silver nitrate and resuspend the cells JCA: Ammend this step. I think you want to say add 2uL of 10mM AgNO3 stock to 200uL of washed cells and vortext to mix (or something like that).
3. Incubate overnight (24-48 hours) at room temperature
4. observe color change and precipitation of colored compound
5. Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)

other 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)
4. Try growing any functional clones in cultures containing the AgNO3