Team:Calgary/Notebook/Protocols/escapersassay

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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>
<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>
<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>
 +
<br>
 +
<li>Colonies were counted to measure CFU.</li>
 +
<br>
 +
<li>Prha-B0034-S7 was transformed into Top 10 and glyA knockout.</li>
 +
<br>
 +
<li>Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.</li>
 +
<br>
 +
<li>The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.</li>
 +
<ul>
 +
<li>M9 minimal media with 0.2% glucose.</li>
 +
<li>M9 minimal media with 0.2% rhamnose.</li>
 +
</ul>
 +
<br>
 +
<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>
 +
<br>
 +
<li>Colonies were counted to measure CFU.</li>
 +
 +
</ol>
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</html>
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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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