Team:Berkeley Wetlab/Passenger: Ag4 Peptide
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AgNO3 Reduction
This assay tests for the presence of the AG4 silver binding peptide on the E. coli cell surface, and it's ability to bind and reduce silver
constructs: AG4 peptide (8) 1363 negative control (1)
experiment was done in triplicate
Initially verified: AgNO3 does not react with LB or TBS
growing cells
- inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
- dilute culture 1:100 into media with arabinose and induce for 5-12hours
- pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
Wash cells and incubate in AgNO3
- pipet 2mls of culture into a 24-well block
- pellet the saturated induced culture
- pour out the supernatant
- wash cells with 200ul of TBS 2X
Treating with Silver
- make 10mM stock of AgNO3
- add 200uL, 1ml, and 2ml of 0.1 mM silver nitrate and resuspend the cells
- incubate overnight at room temperature
- observe color change and precipitate formation
- take pictures with TEM
Cleaning
- Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
- Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL)
- Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
- Add 1 mL of TBS (pH 7.4) and resuspend
- Centrifuge the colony solution for 30 seconds
- Pour out the supernatant
- Add 200uL of TBS and resuspend
- Dilute an aliquot of the solution 10X. Measure OD of solution using spectrophotometer (at 600nm)
Treating with Silver
- Make 10mM stock of AgNO3
- 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).
- Incubate overnight (24-48 hours) at room temperature
- observe color change and precipitation of colored compound
- Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)
other controls
- Chemically reduce silver nitrate to precipitate silver (tells us silver nitrate is precipitating)
- Lyse cells over-expressing AG4 and run assay (tells us if AG4 is precipitating silver)
- Wash non-AG4 cells with silver nitrate and look for colour change (tells us if precipitation occurs without AG4)
- 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