Team:Calgary/Notebook/Protocols/escapersassay

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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.