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Applications of BBa_I14017

User Reviews

UNIQa011dd1fe0ccc13a-partinfo-00000000-QINU

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ETH Zurich 2014

Characterization of two-order crosstalk on the promoter

Background information

System considered

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)[au]
a = basal expression rate [au](“leakiness”)
b = maximum expression rate [au]("full induction")
n = Hill coefficient (“cooperativity”)
K_m = Half-maximal effective concentration (“sensitivity”)
[AHL]=OHHL concentration [nM]

First-order crosstalk

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

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

Other regulatory proteins, like LuxR, bind to different HSL and activates the promoter.

Results

**Table 1** Crosstalk matrix for the promoter prhl (BBa_I14017)
In this set of experiments the promoter pRhl was tested for potential crosstalk. In the top left position we observe the induction of pRhl by C4-HSL bound to the regulator RhlR. The switching behaviour was observed at a C4-HSL concentration of 1 μM. In the case of 3OC12-HSL binding the RhlR regulator and subsequently the promoter pRhlinsignificant crosstalk has been observed. Severe crosstalk was observed in the case of 3OC6-HSL binding the RhlR regulator followed by induction of pRhl. The transition occurred at a concentration of the inducer molecule of 1 μM but compared to the reference curve a lower value of fluorescence per OD was observed (1000 a.u.). Another case of crosstalk with the pRhl was detected with 3OC12-HSL binding to the corresponding LasR regulator followed by inducing the promoter pRhl. Here switching occurred at a concentration 1 nM of 3OC12-HSL and reached fluorescence per OD of 750 a.u.. This is approximately 0.5 fold the value of the fluorescence per OD shown by the reference curve indicated in green.