Team:Washington/Protocols/PUR Assay

From 2012.igem.org


PUR Esterase Assay

  1. Transformed MG1655 with PUR Esterase pGA3K3 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K892012 BBa_K892012]
    • Followed the [https://2012.igem.org/Team:Washington/Protocols/Elect. electroporation protocol]
    • Plated on 200µL of rescue on LB + 1% kanamycin agar plate
  2. Plated MG1655 on LB agar plates
  3. Next day, picked 1 colony from each plate and grew them in 50mL of TB
    • Added kanamycin to the the transformed cell's liquid culture (final concentration of 1% mass/volume)
  4. Next day, spun the 50mL overnight cultures down at 4000g for 10 minutes
  5. Poured out the supernatant
  6. Resuspended cells in DI water to make each culture equal in 1-cm path length OD (we chose and OD of 1.4)
  7. Aliquoted out 1mL from each culture into 3 different microcentrifuge tubes tubes.
  8. Using a sonicator, sonicated the aliquots at an amplitude of 20 with 1 second pulses on and off for 30 seconds.
  9. Applied lysate to pre weighed samples of foam within 25mL culture tubes
  10. Pipetted 2 mL of LB into a 25 mL culture tube with a pre weighed foam sample (negative control)
  11. Repeat steps 7-10 three more times.
  12. Set culture tubes with lysate and foam as well as with the control media onto bench top and leave overnight
  13. Carefully take out foam samples and wash thoroughly with DI water
  14. Leave washed foam samples to dry in 65º C incubator overnight
  15. Weigh dried foam samples

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