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

  1. inoculate cells from stock into LB with the appropriate antibiotics and grow to saturation (12+ hours)
  2. dilute culture 1:100 into media with arabinose and induce for 5-12hours
  3. pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD

Wash cells and incubate in AgNO3

  1. pipet 2mls of culture into a 24-well block
  2. pellet the saturated induced culture
  3. pour out the supernatant
  4. wash cells 2X with 200ul of TBS

Treating with Silver

  1. make 10mM stock of AgNO3
  2. add 200uL, 1ml, and 2ml of 0.1 mM silver nitrate and resuspend the cells (add 180 ul of TBS to each pellet, resuspend cells, add 20 ul of 1 mM AgNO3)
  3. incubate overnight at room temperature
  4. observe color change and precipitate formation
  5. take pictures with TEM

TBS recipe

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