Difference between revisions of "Part:BBa R0077:Experience"

Revision as of 23:42, 5 October 2011

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Applications of BBa_R0077

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UNIQ13b5281fd5e732e4-partinfo-00000000-QINU UNIQ13b5281fd5e732e4-partinfo-00000001-QINU First, we induced generator cell with 10-3M IPTG for 16 hours, then centrifuged cells and collect the supernatant. We use this supernatant as original 3OH-C14:1-HSL solution，then we diluted it to 10-7, 5×10-7, 10-6, 5×10-6, 10-5, 6×10-5, 8×10-5, 10-4, 2×10-4, 4×10-4, 7×10-4, 10-3, 3×10-3, 6×10-3, 8×10-3, 10-2 of original solution concentration respectively. Using these diluted supernatants to induce receiver cells (OD600 0.4) at 37℃ for 3 hours, then centrifuged cells and resuspended in phosphate buffer solution (PBS). The GFP fluorescence of each culture was obtained using enzyme-labeled assay. The result is shown in figure 6.

Figure 6. Dose Response Curve. Vertical axis RFU/OD represents GFP expression measured through enzyme-labeled assay and the Horizontal axis represent different relative dilution concentration of 3OH-C14:1-HSL. Data was fitted with Hill function.

salicylate system

We cultivate the recevier E.coli until the OD up to 0.4 and add salicylateinto 16 EP tube containing aforementioned E.coli respectively. Different concentration of salicylate solution will be added, that is 10-7 M, 10-6 M, 5×10-6 M, 10-5 M, 2.5×10-5 M, 5×10-5 M, 7.5×10-5 M, 10-4 M, 2.5×10-4 M, 5×10-4 M, 7.5×10-4 M, 10-3 M. Cultivate the cell for 3 hours at 37℃. Then centrifuged cells and resuspended in phosphate buffer solution (PBS). The GFP fluorescence of each culture was obtained using FCM(flow cytometry). The result is shown in figure 7.

Figure 7. Dose response curve. Horizontal axis represents GFP expression measured by flow cytometry and the vertical axis represen different salicylate concentration. Data was fitted with Hill function.

• Hill function:>

Figure 8. The modeling parameter of dose response curve of salicylate system and cin system. Dose response curve data was fiitted by Hill fuction.