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Team:Calgary/Notebook/Protocols/escapersassay
From 2012.igem.org
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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.
- Prha-B0034-S7 was transformed into Top 10 and glyA knockout.
- Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.
- 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.
- 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.
- Colonies were counted to measure CFU.
- Prha-B0034-S7 was transformed into Top 10 and glyA knockout.
- Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.
- 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.
- 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.
- Colonies were counted to measure CFU.
