Team:British Columbia/Protocols/ConsortiaFluor

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<h1>Consortia Fluorescence Monitoring</h1>
<h1>Consortia Fluorescence Monitoring</h1>
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1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin .  
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#Grow overnight 5mL cultures of auxotrophs(MetA<sup>-</sup>, TrpA<sup>-</sup>, and TyrA<sup>-</sup>)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .  
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#Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.  
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2. Spin down cells using a centrifuge (1600g, 10min). Pour out LB supernatant.  
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#Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
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#Perform step 3 three times.  
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3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600g, 10min)
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#Resuspend in 5 mL M9 Media.
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#Measure the respective O.D 600 with a spectrometer.
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4. Perform step 3 three times.  
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#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).
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#Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.
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5. Resuspend in 5mL M9 Media.
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6. Measure the respective O.D 600 with a spectrometer.
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7. Inoculate in a 96 well plate containing 200uL
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. Incubate at 37°C shaking at 200rpm.
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4. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
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5.  and extract 25mL of supernatant.
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6. Acidify supernatant to pH of 2.0 with 6N HCl to inactivate enzymes.
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7. Extract with equal volume of ethyl acetate.
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<h1>Excitation and Emission Wavelengths of Fluorescent Proteins</h1>
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8. Dry extract with nitrogen gas and re-suspend in mobile phase with 80% acetonitrile.
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*EYFP - Excitation: 514nm    Emission: 527nm
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9. Filter extract resuspended in acetonitrile using reverse phase chromatography and C-18 column.
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*ECFP - Excitation: 439nm    Emission: 476nm
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10. Elute mobile phase at a flow rate of 0.8 ml/min and peaks of DBT were detected at 280nm by HPLC.
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*ERFP - Excitation: 584nm    Emission: 607nm

Latest revision as of 03:42, 4 October 2012

British Columbia - 2012.igem.org
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
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