Difference between revisions of "Part:BBa C0179:Experience"

(→‎Modeling crosstalk)
(→‎Modeling crosstalk)
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[[File:ETHZ_HillEq.png|center|200px]]
 
[[File:ETHZ_HillEq.png|center|200px]]
 
<p>The fitting of the graphs was performed using the following equation :<br><br>
 
<p>The fitting of the graphs was performed using the following equation :<br><br>
−
rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[au]<br>
+
rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.]<br>
−
a    = basal expression rate [au](“leakiness”)<br>
+
a    = basal expression rate [a.u.](“leakiness”)<br>
−
b = maximum expression rate [au]("full induction")<br>
+
b = maximum expression rate [a.u.]("full induction")<br>
 
n      = Hill coefficient (“cooperativity”)<br>
 
n      = Hill coefficient (“cooperativity”)<br>
 
K<sub>m</sub>  = Half-maximal effective concentration (“sensitivity”)<br>
 
K<sub>m</sub>  = Half-maximal effective concentration (“sensitivity”)<br>
Line 54: Line 54:
 
|-
 
|-
 
! [https://parts.igem.org/Part:BBa_R0079 Plas]
 
! [https://parts.igem.org/Part:BBa_R0079 Plas]
−
|a = 5.274 (0, 15.51) [au]<br> n = 11.67 (0, 3.763e8)<br> Km = 1.396 (0, 1.504e7) [nM]<br>b = 189 (162.5, 215.6) [au]<br>
+
|a = 5.274 (0, 15.51) [a.u.]<br> n = 11.67 (0, 3.763e8)<br> Km = 1.396 (0, 1.504e7) [nM]<br>b = 189 (162.5, 215.6) [a.u.]<br>
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk
 
|-
 
|-
 
! [https://parts.igem.org/Part:BBa_R0062 Plux]
 
! [https://parts.igem.org/Part:BBa_R0062 Plux]
−
| a = 362.4 (158.2,566.6) [au]<br> n = 0.7898 (0.3241,1.256)<br>Km = 1.269 (0, 2.548) (nM)<br> b = 1.253e4 (1.085e4, 1.422e4) [au]<br>
+
| a = 362.4 (158.2,566.6) [a.u.]<br> n = 0.7898 (0.3241,1.256)<br>Km = 1.269 (0, 2.548) [nM]<br> b = 1.253e4 (1.085e4, 1.422e4) [a.u.]<br>
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk
 
|-
 
|-
 
! [https://parts.igem.org/Part:BBa_I14017 Prhl]
 
! [https://parts.igem.org/Part:BBa_I14017 Prhl]
−
| a = 149.3 (140.6, 158.1) [au]<br>n = 1.366 (0.808, 1.923) <br> Km = 1.674 (1.259, 2.09) (nM)<br> b = 628.9 (599, 658.7) [au]<br>
+
| a = 149.3 (140.6, 158.1) [a.u.]<br>n = 1.366 (0.808, 1.923) <br> Km = 1.674 (1.259, 2.09) [nM]<br> b = 628.9 (599, 658.7) [a.u.]<br>
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk
 
| No crosstalk

Revision as of 12:57, 24 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_C0179

User Reviews

UNIQ99d3a14f3ff4263f-partinfo-00000000-QINU


••••

ETH Zurich 2014

Characterization of two-order crosstalk

Background information

System considered

Modeling crosstalk

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

ETHZ HillEq.png

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”)
Km = Half-maximal effective concentration (“sensitivity”)
[AHL] = AHL concentration [nM]


Parameters of Hill Function for crosstalk with LasR (with 95% confidence bounds)
3OC12-HSL 3OC6-HSL C4-HSL
Plas a = 5.274 (0, 15.51) [a.u.]
n = 11.67 (0, 3.763e8)
Km = 1.396 (0, 1.504e7) [nM]
b = 189 (162.5, 215.6) [a.u.]
 No crosstalk No crosstalk
Plux a = 362.4 (158.2,566.6) [a.u.]
n = 0.7898 (0.3241,1.256)
Km = 1.269 (0, 2.548) [nM]
b = 1.253e4 (1.085e4, 1.422e4) [a.u.]
 No crosstalk No crosstalk
Prhl a = 149.3 (140.6, 158.1) [a.u.]
n = 1.366 (0.808, 1.923)
Km = 1.674 (1.259, 2.09) [nM]
b = 628.9 (599, 658.7) [a.u.]
 No crosstalk No crosstalk

First-order crosstalk

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

ETH Zurich 1crosstalkPlas.png

Second Level crosstalk: LasR binds to 3OC12-HSL, its natural HSL, and activates different promoter

400px

Second order crosstalk: Combination of both cross-talk levels

Results

Table 1 Crosstalk matrix for the regulator LasR (BBa_C0179)

In all the measurements conducted to create this matrix the regulator LasR was the basis and was induced in six different variations shown.

ETH Zurich 2014 qs-table Corner LasR.png ETH Zurich 2014 qs-table 3OC12-HSL.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table C4-HSL.png
ETH Zurich 2014 qs-table pLas.png ETH Zurich 2014 qs-table PlasRef.png ETH Zurich 2014 qs-table PlasLasRLuxAHL.png ETH Zurich 2014 qs-table PlasLasRRhlAHL.png
ETH Zurich 2014 qs-table pLux.png ETH Zurich 2014 qs-table PluxLasRLasAHL.png ETH Zurich 2014 qs-table PluxLasRLuxAHL.png ETH Zurich 2014 qs-table PluxLasRRhlAHL.png
ETH Zurich 2014 qs-table pRhl.png ETH Zurich 2014 qs-table PrhlLasRLasAHL.png ETH Zurich 2014 qs-table PrhlLasRLuxAHL.png ETH Zurich 2014 qs-table PrhlLasRRhlAHL.png



Antiquity

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

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 RhlR (C0171) continuously in our system. BBa K575032


•••••

iGEM Dundee 2014

Dundee iGEM 2014 used this lasR coding sequence to build two composite parts termed BBa_K1315009 and BBa_K1315010. These were designed as biosensors for Pseudomonas aeruginosa AutoInducer-1 (PAI-1), and were to be used in a bio-electronic device to improve diagnostics for Cystic Fibrosis patients. Details of experimental work are logged on the experience pages of BBa_K1315009 and BBa_K1315010.