Team:Calgary/Notebook/Protocols/escapersassay

From 2012.igem.org

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TITLE=Escaper assay for the rhamnose system|
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<p> Three glycine assays were performed in order to better characterize the <i>glyA</i> Keio Knockout Strain (JW2535-1). These included an assay to determine the minimal amount of glycine required for survival of the auxotroph in minimal media which was used in previous Petrobrick assays. Additionally, the functionality of the Petrobrick was evaluated in this strain to see if similar concentrations of hydrocarbons could be produced as in WT strains of <i>E. coli</i>. In order to evaluate if this strain could be used in conjunction with our kill switch mechanisms, we used these systems in conjunction.
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<p>The following assay is based on CFU in both Top 10 and glyA knockout of <i>E. coli</i>.</p>
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<ol>
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<li>Prha-B0034-S7 was transformed into Top 10 and glyA knockout.</li>
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<br>
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<li>Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.</li>
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<br>
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<li>The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.</li>
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<ul>
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<li>M9 minimal media with 0.2% glucose.</li>
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<li>M9 minimal media with 0.2% rhamnose.</li>
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</ul>
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<br>
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<h2>Media Optimization for the Glycine Auxotroph</h2>
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<li>All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.</li>
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<br>
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<li>Colonies were counted to measure CFU.</li>
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<br>
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<li>Prha-B0034-S7 was transformed into Top 10 and glyA knockout.</li>
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<br>
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<li>Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.</li>
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<br>
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<li>The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.</li>
<ul>
<ul>
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<li>The <i>glyA</i> auxotroph was grown in LB media overnight at 37<sup>o</sup>C with shaking.
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<li>M9 minimal media with 0.2% glucose.</li>
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<li>Cells were washed in M9 minimal media two times and spun in between at 4,000 RPM at 4<sup>o</sup>C for 10 min. each time.
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<li>M9 minimal media with 0.2% rhamnose.</li>
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<li> Finally, washed cells were subcultured into 5 mL of M9 minimal media supplemented with 1% (w/v) glucose and 50 ug/mL Kanamycin.
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<li>Cells were grown at 37<sup>o</sup>C with shaking for 12 and 24 hours and OD<sub>600</sub> measurements were used to quantitate growth.
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</ul>
</ul>
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<br>
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<li>All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.</li>
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<br>
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<li>Colonies were counted to measure CFU.</li>
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<h2>Glycine Auxotrophy Effect on Petrobrick Output</h2>
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</ol>
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<p>The effect of the auxotroph on the output of the Petrobrick was determined as discussed in the flux balance analysis assay using 50 mM glycine. <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Modelvalidation">Click here</a> to view this protocol.
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Latest revision as of 03:41, 27 October 2012

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Escaper assay for the rhamnose system

The following assay is based on CFU in both Top 10 and glyA knockout of E. coli.

  1. Prha-B0034-S7 was transformed into Top 10 and glyA knockout.

  2. Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.

  3. The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.
    • M9 minimal media with 0.2% glucose.
    • M9 minimal media with 0.2% rhamnose.

  4. All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.

  5. Colonies were counted to measure CFU.

  6. Prha-B0034-S7 was transformed into Top 10 and glyA knockout.

  7. Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.

  8. The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.
    • M9 minimal media with 0.2% glucose.
    • M9 minimal media with 0.2% rhamnose.

  9. All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.

  10. Colonies were counted to measure CFU.

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