Team:Calgary/Notebook/Protocols/escapersassay

From 2012.igem.org

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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>
<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>
 +
<li>Colonies were counted to measure CFU.</li>
 +
 +
<li>Prha-B0034-S7 was transformed into Top 10 and glyA knockout.</li>
 +
 +
<li>Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.</li>
 +
 +
<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>
 +
 +
<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>
 +
 +
<li>Colonies were counted to measure CFU.</li>
</ol>
</ol>

Revision as of 03:39, 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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