Template:SBB-Protocols Assay3: Difference between revisions

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=== Procedure ===
=== Procedure ===
==Incubating Colonies==
 
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min
==Picking and Incubating Colonies==
#Plate on selective antibiotics, let incubate overnight
==Picking Colonies==
*For each construct you will pick and later miniprep 2 colonies
*Add 4mL of LB media with the appropriate antibiotics to a clean test tube
*Add 4mL of LB media with the appropriate antibiotics to a clean test tube
*Pick a well-isolated, round, and "normal" looking colony with a toothpick
*Pick a well-isolated, round, and "normal" looking colony of our proposed silver binding peptide with a toothpick
*Drop it in the test tube
*Drop it in the test tube
*Incubate at 37 overnight
*Incubate at 37 overnight
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#Centrifuge the colony solution for 30 seconds
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
#Add 50 uL of Tris buffer
#Add 200uL of 0.1 mM  silver nitrate and resuspend the cells
#Incubate at 37 overnight
==Further Cleaning==
#Notice the color of the solution after incubation
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
#Add  200uL of Tris Buffer and resuspend
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant
#Add  200uL of Tris Buffer and resuspend


==Control Group==


# Wash with Tris buffer
==Picking and Incubating Colonies==
# Incubate in 0.1 mM silver nitrate for 24-48h at room temperature
*Add 4mL of LB media with the appropriate antibiotics to a clean test tube
# Centrifuge the solution
*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==
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
#Add  200uL of 0.1 mM silver nitrate and resuspend the cells
#Incubate at 37 overnight
==Further Cleaning==
#Notice the color of the solution after incubation
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
#Add  200uL of Tris Buffer and resuspend
#Centrifuge the colony solution for 30 seconds
#Pour out the supernatant
#Add  200uL of Tris Buffer and resuspend
# Look for reddish colour change
# Look for reddish colour change


Revision as of 11:21, 15 April 2009

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 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

Control Group

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

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)
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