Difference between revisions of "Part:BBa R0063:Experience"

Latest revision as of 14:41, 30 October 2013

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Applications of BBa_R0063

The stochastic dynamics of the constitutive promoter family by 2013 Fudan iGEM team

We fused the constitutive promoters to a sfGFP reporter to test their stochastic dynamics by means of measuring fluorescence intensity of sfGFP using flow cytometry. In the meantime, we inserted the 16nt Csy4 insulator between the promoter and the reporter to stardardize the expression of the reporter downstream by eliminating the influence from 5'UTR. The following chart shows the distribution of sfGFP expression in the circumstance of promoter R0063.

Figure 1. The distribution of the sfGFP expression tested by flow cytometry with promoter R0063 and Csy4 insulator.

Figure 2. The intensity of different promoters is shown by the column height and the randomness of their functions by the bar width.

this part was used by ETH Zurich-iGEM 2013
The P_LuxL was used as a part of a positive feedback loop to improve the leakiness of an AHL induced reporter system based on BBa_J09855 and GFP BBa_E0840 expressed from P_LuxR BBa_R0062. Even in the absence of AHL we could observe GFP expression. Assuming that the basal expression comes from an AHL-independent activation of P_LuxR through LuxR we expressed LacI BBa_J24679 from the BBa_R0063 P_LuxL promoter. Testing the constructs in liquid culture over 16 h without and with induction through AHL (100 nM) showed a significant improvement of the On/Off ratio.

Figure 3. Usage of a P_LuxL-LacI feedback to reduce basal expression of GFP.

User Reviews

UNIQf1fba75541088a58-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

UNIQf1fba75541088a58-partinfo-00000002-QINU

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 this part was used by <b>ETH Zurich-iGEM 2013</b>
-<br> The P<sub>LuxL</sub> was used as a part of a positive feedback loop to improve the leakiness of an AHL induced reporter system based on [https://parts.igem.org/Part:BBa_J09855 BBa_J09855] and GFP [https://parts.igem.org/Part:BBa_E0840 BBa_E0840] expressed from P<sub>LuxR</sub> [https://parts.igem.org/Part:BBa_R0062 BBa_R0062]. Even in the absence of AHL we could observe GFP expression. Assuming that the basal expression comes from an AHL-independent activation of P<sub>LuxR</sub> through LuxR we expressed LacI [https://parts.igem.org/Part:BBa_J24679 BBa_J24679] from the BBa_R0063 P<sub>LuxL</sub> promoter. Testing the constructs in liquid culture over 16 h without and with induction of AHL (100 nM) showed a significant improvement of the On/Off ratio.
+<br> The P<sub>LuxL</sub> was used as a part of a positive feedback loop to improve the leakiness of an AHL induced reporter system based on [https://parts.igem.org/Part:BBa_J09855 BBa_J09855] and GFP [https://parts.igem.org/Part:BBa_E0840 BBa_E0840] expressed from P<sub>LuxR</sub> [https://parts.igem.org/Part:BBa_R0062 BBa_R0062]. Even in the absence of AHL we could observe GFP expression. Assuming that the basal expression comes from an AHL-independent activation of P<sub>LuxR</sub> through LuxR we expressed LacI [https://parts.igem.org/Part:BBa_J24679 BBa_J24679] from the BBa_R0063 P<sub>LuxL</sub> promoter. Testing the constructs in liquid culture over 16 h without and with induction through AHL (100 nM) showed a significant improvement of the On/Off ratio.
 [[File:ETHZ_registryentryR0063.png|800px|thumb|center|'''Figure 3'''. Usage of a P<sub>LuxL</sub>-LacI feedback to reduce basal expression of GFP.]]