Team:British Columbia/Protocols/ConsortiaFluor


British Columbia -
UBC iGEM 2012 protocols

Consortia Fluorescence Monitoring

  1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .
  2. Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.
  3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
  4. Perform step 3 three times.
  5. Resuspend in 5 mL M9 Media.
  6. Measure the respective O.D 600 with a spectrometer.
  7. Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200 μL M9 with ampicillin (100 mg/mL).
  8. Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.

Excitation and Emission Wavelengths of Fluorescent Proteins

  • EYFP - Excitation: 514nm Emission: 527nm
  • ECFP - Excitation: 439nm Emission: 476nm
  • ERFP - Excitation: 584nm Emission: 607nm