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

Growing Cells in LB, AgNO3, TBS

wash cells with TBS and resuspend it
grow a portion of the cells in 3ml LB
add 0.2ml TBS
add 0.1mM AgNO3
grow cells overnight
hope that AgNO3 does not kill the cells and a color change will occur
one sample (11) also has arabinose in it to test for a difference in expression

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

Procedure carried out on 22 April 2009

Preparation of Tris buffer solution

• Add 605 mg Tris and 876 mg NaCl to 80 ml H2O
• Adjust pH to 7.4 by adding HCl and bring volume to 100 ml

Preparation of AgNO3 solution

• Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
• AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
• Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O

Preparation of cells for assay

• Spin down 2ml of cells and throw away supernatant
• Add 200 ul of TBS and resuspend cells
• Spin down the cells and throw away supernatant

Preparing cells for growth in LBTAg media

• Dispense 3ml of LB media into 9 chambers in growth rack
• Add 200 ul of Tris buffer solution to the LB media
• Add 32 ul of 10 mM AgNO3 to make 0.1 mM LBTAg media
• Pick cells from each washed pellet (above) and add to corresponding growth chambers
  • 11-17 + Control + another one of 11
• Add 3ul of 1000x Arabinose to the 9th chamber containing a second culture of cells 11
• Cover the rack and incubate overnight at 37C

Running the assay on cells

• Add 180 ul of TBS to each pellet (prepared above)
• Resuspend cells
• Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
• Incubate overnight at room temperature and with agitation