Team:British Columbia/Protocols/ConsortiaFluor

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<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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2. Spin down cells using a centrifuge (1600g, 10min). Pour out LB supernatant.
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3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600g, 10min)
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4. Perform step 3 three times.
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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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8. Dry extract with nitrogen gas and re-suspend in mobile phase with 80% acetonitrile.
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9. Filter extract resuspended in acetonitrile using reverse phase chromatography and C-18 column.
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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.

Revision as of 23:22, 3 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 .

2. Spin down cells using a centrifuge (1600g, 10min). Pour out LB supernatant.

3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600g, 10min)

4. Perform step 3 three times.

5. Resuspend in 5mL M9 Media.

6. Measure the respective O.D 600 with a spectrometer.

7. Inoculate in a 96 well plate containing 200uL


. Incubate at 37°C shaking at 200rpm.

4. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.

5. and extract 25mL of supernatant.

6. Acidify supernatant to pH of 2.0 with 6N HCl to inactivate enzymes.

7. Extract with equal volume of ethyl acetate.

8. Dry extract with nitrogen gas and re-suspend in mobile phase with 80% acetonitrile.

9. Filter extract resuspended in acetonitrile using reverse phase chromatography and C-18 column.

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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