Team:Calgary/Notebook/Protocols/Modelvalidation

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<p> After running the model for the pathway in question, the PetroBrick (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K590025">BBa_K590025</a></b>), overnight cultures were made adding different combinations of metabolites found to increase product output from the model. </p>
<p> After running the model for the pathway in question, the PetroBrick (<b><a href="http://partsregistry.org/wiki/index.php?title=BBa_K590025">BBa_K590025</a></b>), overnight cultures were made adding different combinations of metabolites found to increase product output from the model. </p>
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<li>PetroBrick culture was grown in 3x 20mL cultures from isolated plate colonies supplemented with 50 ug/mL chloramphenicol.  These cultures were grown overnight at 37<sup>o</sup>C with shaking.
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<li>Culture tubes had 5 mL of minimal <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 media</a> with 1% (w/v) filter sterilized glucose, and 50 ug/mL chloramphenicol added to each tube.
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<li>All chemicals added were at a concentration of 50 mM except for AMP (100 mg/L) and L-aspartate (100mg/L). Stocks were dissolved in water at concentrations ~100x what was being added (provided that the compound was soluble) to ensure that changes in volume would not account for differences seen.
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<li>Cells were allowed to grow for 72 hours at 37<sup>o</sup>C with shaking.
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<li>Cultures then had OD<sub>600</sub> readings taken to normalize against.
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<li>Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min.
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<li>1 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4<sup>o</sup>C
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<li>250 uL of the ethyl acetate layer was ran onto the <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">GC-MS</a>.
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Latest revision as of 00:11, 27 October 2012

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Modelling Validation Experiments

After running the model for the pathway in question, the PetroBrick (BBa_K590025), overnight cultures were made adding different combinations of metabolites found to increase product output from the model.

  1. PetroBrick culture was grown in 3x 20mL cultures from isolated plate colonies supplemented with 50 ug/mL chloramphenicol. These cultures were grown overnight at 37oC with shaking.
  2. Culture tubes had 5 mL of minimal M9 media with 1% (w/v) filter sterilized glucose, and 50 ug/mL chloramphenicol added to each tube.
  3. All chemicals added were at a concentration of 50 mM except for AMP (100 mg/L) and L-aspartate (100mg/L). Stocks were dissolved in water at concentrations ~100x what was being added (provided that the compound was soluble) to ensure that changes in volume would not account for differences seen.
  4. Cells were allowed to grow for 72 hours at 37oC with shaking.
  5. Cultures then had OD600 readings taken to normalize against.
  6. Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min.
  7. 1 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4oC
  8. 250 uL of the ethyl acetate layer was ran onto the GC-MS.

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