Template:SBB-Protocols Assay3

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=== Procedure ===
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== Procedure ==
===Picking and Incubating Colonies===
===Picking and Incubating Colonies===

Revision as of 14:28, 15 April 2009

Contents

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.

Materials

Procedure

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 our proposed silver binding peptide with a pipet tip
  • Drop it in the test tube
  • Incubate at 37 overnight

Cleaning

  1. Notice the color of the solution after incubation
  2. Centrifuge the colony solution for 30 seconds
  3. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
  4. Add 200uL of Tris Buffer and resuspend
  5. Centrifuge the colony solution for 30 seconds
  6. Pour out the supernatant
  7. Add 200uL of Tris Buffer and resuspend

Treating with Silver

  1. Centrifuge the colony solution for 30 seconds
  2. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
  3. Add 200ul of PBS, centrifuge for 30 seconds, and throw out the supernatant
  4. Add 200uL of 0.1 mM silver nitrate and resuspend the cells
  5. Incubate at 37 overnight


Control Group

  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)

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

Treating with Silver

  1. Centrifuge the colony solution for 30 seconds
  2. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
  3. Add 200uL of 0.1 mM silver nitrate and resuspend the cells
  4. Incubate at 37 overnight

Further Cleaning

  1. Notice the color of the solution after incubation
  2. Centrifuge the colony solution for 30 seconds
  3. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
  4. Add 200uL of Tris Buffer and resuspend
  5. Centrifuge the colony solution for 30 seconds
  6. Pour out the supernatant
  7. Add 200uL of Tris Buffer and resuspend
  8. Look for reddish colour change
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