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

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=== Modeling crosstalk ===
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Each experimental data set was fitted to an Hill function using the Least Absolute Residual method.
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[[File:ETHZ_HillEq.png|center|200px]]
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<p>The fitting of the graphs was performed using the following equation :<br><br>
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rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.]<br>
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a    = basal expression rate [a.u.](“leakiness”)<br>
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b = maximum expression rate [a.u.]("full induction")<br>
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n      = Hill coefficient (“cooperativity”)<br>
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K<sub>m</sub>  = Half-maximal effective concentration (“sensitivity”)<br>
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[AHL]= AHL concentration [nM]</p>
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<br clear="all"/>
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{| border="1" class="wikitable" style="margin: 1em auto 1em auto;text-align:center;"
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|+ Parameters of HillFunction for crosstalk with RhlR (with 95% confidence bounds)
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!
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! [[AHL|C4-HSL]]
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! [[3OC6HSL|3OC6-HSL]]
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! [[AHL|3OC12-HSL]]
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|-
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! [https://parts.igem.org/Part:BBa_I14017 Prhl]
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| a = 178.4 (174.9, 182) [a.u.] <br> n = 1.053 (0.9489, 1.157)<br> Km = 1969 (1625, 2313) [nM]<br>b = 1736 (1629, 1842) [a.u.]<br>
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| a = 169.1 (155.2, 182.9) [a.u.]<br> n = 0.507 (0.2303, 0.7837) <br> Km = 1.08e8(0, 2.681e10) [nM]<br> b = 9.708e4 (0, 1.192e7) [a.u.]<br>
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| a = 162.8 (150.4, 175.1) [a.u.]<br> n = 0.404 (0, 0.998)<br> Km = 9.627e8 (0, 7.824e11) [nM]<br>b = 2.537e4 (0, 8.109e6) [a.u.]<br>
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|-
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! [https://parts.igem.org/Part:BBa_R0062 Plux]
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| a = 309.6 (283.6, 335.6) [a.u.]<br> n = 0.741 (0.6463, 0.8357) <br> Km = 2738 (1411, 4064) [nM] <br> b = 1.22e4 (1.045e4,1.45e4) [a.u.] <br>
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| a = 313.2 (296.8, 329.5) [a.u.]<br> n = 0.9427 (0.7565, 1.129)<br> Km = 1210 (565, 1855) [nM] <br> b = 8361 (7001, 9722) [a.u.] <br>
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| No crosstalk
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|-
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! [https://parts.igem.org/Part:BBa_R0079 Plas]
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| No crosstalk
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| No crosstalk
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| No crosstalk
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|}
  
 
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Revision as of 13:48, 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_C0171

User Reviews

UNIQ8e21568612984e32-partinfo-00000000-QINU


••••

ETH Zurich 2014

Characterization of two-order crosstalk on the promoter

Background information

First-order crosstalk

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

ETH Zurich 1crosstalkPrhl.png

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

ETH Zurich 2crosstralkRrhl.png

Second order crosstalk: Combination of both cross-talk levels

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.

ETH Zurich 2014 qs-table Corner RhlR.png ETH Zurich 2014 qs-table C4-HSL.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table 3OC12-HSL.png
ETH Zurich 2014 qs-table pRhl.png ETH Zurich 2014 qs-table PrhlRef.png ETH Zurich 2014 qs-table PrhlRhlRLuxAHL.png ETH Zurich 2014 qs-table PrhlRhlRLasAHL.png
ETH Zurich 2014 qs-table pLux.png ETH Zurich 2014 qs-table PluxRhlRRhlAHL.png ETH Zurich 2014 qs-table PluxRhlRLuxAHL.png ETH Zurich 2014 qs-table PluxRhlRLasAHL.png
ETH Zurich 2014 qs-table pLas.png ETH Zurich 2014 qs-table PlasRhlRRhlAHL.png ETH Zurich 2014 qs-table PlasRhlRLuxAHL.png ETH Zurich 2014 qs-table PlasRhlRLasAHL.png

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 HillFunction 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.

UNIQ8e21568612984e32-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