Team:British Columbia/Protocols/ConsortiaFluor
From 2012.igem.org
Consortia Fluorescence Monitoring
- Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .
- Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.
- Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
- Perform step 3 three times.
- Resuspend in 5 mL M9 Media.
- Measure the respective O.D 600 with a spectrometer.
- 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).
- 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