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

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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.
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<I>Peking_S iGEM 2011</I>
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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 1.
  
 
<center>[[Image:Refigure1.png‎]]</center>
 
<center>[[Image:Refigure1.png‎]]</center>
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''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.
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''Figure 1. 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.
  
 
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salicylate system
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Time Dependence
  
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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.
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We induced E.coli with receiver plasmid using supernatant diluted 10-2 times, and measure OD and absorbance of GFP every 15 minutes.
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<center>[[Image:LM9.png]]</center>
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''Figure 2. Time response curve of cin receiver system. We measured RFU and OD600 every 15 minutes and experiment performed in 3 hours.''
  
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<center>[[Image:Refigure2.png]]</center>
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Coordinatiblity
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''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.''
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<center>[[Image:LM12a.png|600px]]</center>
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''Figure 3. Cin receiver system cell distribution of GFP fluorescence intensity.''
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<center>[[Image:Refigure.png|600px]]</center>
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Latest revision as of 19:59, 18 October 2011

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No review score entered. Peking_S iGEM 2011

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 1.

Refigure1.png

Figure 1. 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.

Time Dependence

We induced E.coli with receiver plasmid using supernatant diluted 10-2 times, and measure OD and absorbance of GFP every 15 minutes.

LM9.png

Figure 2. Time response curve of cin receiver system. We measured RFU and OD600 every 15 minutes and experiment performed in 3 hours.

Coordinatiblity

LM12a.png

Figure 3. Cin receiver system cell distribution of GFP fluorescence intensity.

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