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Team:Calgary/Notebook/Protocols/Modelvalidation
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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. | <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. | ||
| - | <li>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. | + | <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. |
<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. | <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. | ||
<li>Cells were allowed to grow for 72 hours at 37<sup>o</sup>C with shaking. | <li>Cells were allowed to grow for 72 hours at 37<sup>o</sup>C with shaking. | ||
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<li>Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min. | <li>Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min. | ||
<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 | <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 | ||
| - | <li>250 uL of the ethyl acetate layer was ran onto the | + | <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.
- 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.
- 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.
- 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.
- Cells were allowed to grow for 72 hours at 37oC with shaking.
- Cultures then had OD600 readings taken to normalize against.
- Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min.
- 1 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4oC
- 250 uL of the ethyl acetate layer was ran onto the GC-MS.
