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Applications of BBa_C0171

User Reviews

UNIQ3e2f2de8e4e8d01d-partinfo-00000000-QINU

••••

ETH Zurich 2014

Characterization of two-order crosstalk on the promoter

Background information

Here, we focus on the characterization of crosstalk of BBa_C0171 RhlR with different AHLs and further crosstalk of RhlR-C4-HSL with the three promoters - pLux, pLas, and pRhl. In the following, we describe all the different levels of crosstalk we have assessed.

First-order crosstalk

In the first order crosstalk section we describe crosstalk of pRhl due to RhlR binding to inducers different from C4-HSL or pRhl itself binding a regulator-inducer pair different from RhlR-C4-HSL.

First Level crosstalk: RhlR binds to different HSL and activates the promoter

In the conventional system C4-HSL binds to its corresponding regulator, RhlR, and activates the pRhl promoter (figure 2, green). However, RhlR can potentially also bind other AHLs and then activate pRhl (figure 2, 3OC12-HSL in red and 3OC6-HSL in blue).

Second Level crosstalk: other regulatory proteins, like LuxR, bind to their natural HSL substrate and activates the promoter

Second order crosstalk: Combination of both cross-talk levels

RhlR can bind to it's native or other AHLs and activates three promoters.

Results

**Table 1** Crosstalk matrix for the regulator LasR (BBa_C0171)
In all the measurements conducted to create this matrix the regulator RhlR was the basis and was induced in six different variations shown.

Modeling crosstalk

Each experimental data set was fitted to an Hill function using the Least Absolute Residual method.

The fitting of the graphs was performed using the following equation :

rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.]
a = basal expression rate [a.u.](“leakiness”)
b = maximum expression rate [a.u.]("full induction")
n = Hill coefficient (“cooperativity”)
K_m = Half-maximal effective concentration (“sensitivity”)
[AHL]= AHL concentration [nM]

Parameters of Hill function for crosstalk with RhlR (with 95% confidence bounds)
	C4-HSL	3OC6-HSL	3OC12-HSL
Prhl	a = 178.4 (174.9, 182) [a.u.] n = 1.053 (0.9489, 1.157) Km = 1969 (1625, 2313) [nM] b = 1736 (1629, 1842) [a.u.]	a = 169.1 (155.2, 182.9) [a.u.] n = 0.507 (0.2303, 0.7837) Km = 1.08e8(0, 2.681e10) [nM] b = 9.708e4 (0, 1.192e7) [a.u.]	a = 162.8 (150.4, 175.1) [a.u.] n = 0.404 (0, 0.998) Km = 9.627e8 (0, 7.824e11) [nM] b = 2.537e4 (0, 8.109e6) [a.u.]
Plux	a = 309.6 (283.6, 335.6) [a.u.] n = 0.741 (0.6463, 0.8357) Km = 2738 (1411, 4064) [nM] b = 1.22e4 (1.045e4,1.45e4) [a.u.]	a = 313.2 (296.8, 329.5) [a.u.] n = 0.9427 (0.7565, 1.129) Km = 1210 (565, 1855) [nM] b = 8361 (7001, 9722) [a.u.]	No crosstalk
Plas	No crosstalk	No crosstalk	No crosstalk

Antiquity

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

UNIQ3e2f2de8e4e8d01d-partinfo-00000003-QINU

No review score entered. Northwestern 2011

The 2011 Northwestern iGEM team used this part as a part of our Pseudomonas Aeruginosa biosensor. We were able to successfully express LasR (C0171) continuously in our system. BBa K575024

@@ Line 47: / Line 47: @@
 == Second order crosstalk: Combination of both cross-talk levels  ==
+RhlR can bind to it's native or other [[AHL|AHLs]] and activates three promoters.
 == Results ==

Difference between revisions of "Part:BBa C0171:Experience"