Difference between revisions of "Part:BBa C0160"
Line 17: Line 17:
 
<!-- -->
 
<!-- -->
  
−
===Further characterization===
+
<h1>Characterization by ETH Zurich 2015</h1>
−
Check the experience part for the characterization done by ETH Zurich 2015 iGEM team.
+
We wanted to know the effect of constitutively expressed aiiA versus no aiiA upon artificial addition of AHL onto P<sub>lux</sub>.
 +
<h2>Genetic design</h2>
 +
We designed two plasmids:
 +
<ul>
 +
  <li>Medium copy backbone with P<sub>lux</sub>-GFP and BBa_J23118-BBa_B0032-luxR-BBa_B0012</li>
 +
  <li>Medium copy backbone with P<sub>lux</sub>-GFP,  BBa_J23118-BBa_B0032-luxR-BBa_B0012 and BBa_J23114-BBa_B0034-aiiA </li>
 +
</ul>
 +
<h2>Plate reader</h2>
 +
E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (1 µ/mL) and chloramphenicol (0.36 µ/mL) at 37°C and 200 rpm. Cultures were diluted 1:50 in fresh LB with the corresponding antibiotic and transferred to a 96-well plate (200 µL/well). Cultures were grown for 90 min to arrive to exponential phase and then different concentrations of lactate were added. Samples were always made in triplicates and a blank of LB with the corresponding lactate concentration was done. During 7 h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 7 min. The plate was always kept at 37°C. We calculated dose-response curves from the exponential phase of the bacteria.
 +
<h2>Modeling</h2>
 +
Each experimental data set was fitted to a Hill function using the Least Absolute Residual method (Fitting Toolbox in MatLab).
 +
    f(AHL) = al+mu*(AHL/K)^n1/(1+(AHL/K)^n1)
 +
 
 +
Where:
 +
<ul>
 +
    <li>al: leakiness in &micro;M/min</li>
 +
    <li>mu: constant in &micro;M/min</li>
 +
    <li>Km: half-maximum effective concentration (a representation of sensitivity) in &micro;M</li>
 +
    <li>n: Hill coefficient (a representation of cooperativity), unitless</li>
 +
</ul>
 +
 
 +
 
 +
{|class="wikitable"
 +
|-
 +
! !!PresentAii||Absent AiiA (lumped parameters)||Absent AiiA (literature parameters)
 +
|-
 +
! rowspan="4" scope="row" | Coefficient values
 +
| K=1e+06  (fixed at bound)|| K=1e+06  (fixed at bound)||      K =      9.983  (8.929, 11.04)
 +
|-
 +
| al = 6134  (5222, 7046)||al = 5711  (-2.36e+04, 3.502e+04)||al =        2876  (1633, 4119)
 +
|-
 +
| mu =  2e+04  (fixed at bound)||mu = 2e+05  (fixed at bound)|| mu =  3.484e+04  (3.338e+04, 3.63e+04)
 +
|-
 +
| n1 =    1  (fixed at bound)|| n1 =      1.646  (-5.203, 8.494)||n1 =      1.089  (0.8608, 1.317)
 +
|-
 +
!rowspan="5" scope="row"|Goodness to fit
 +
|  sse: 5.2051e+09||sse: 3.8192e+12||          sse: 1.4366e+09
 +
|-
 +
|rsquare: 0.8768||rsquare: -89.3659||rsquare: 0.9986
 +
|-
 +
|fe: 10||dfe: 9||dfe: 7
 +
|-
 +
|adjrsquare: 0.8768||adjrsquare: -99.4066||adjrsquare: 0.9980
 +
|-
 +
|rmse: 2.2815e+04||rmse: 6.5142e+05||rmse: 1.4326e+04
 +
|}
 +
 
 +
[[File:ETH15_NoAiiA.png|thumb|400px|right]]
 +
[[File:ETH15_AiiA.png|thumb|400px|left]]

Revision as of 17:49, 17 September 2015

autoinducer inactivation enzyme aiiA (no LVA)

same as Part:BBa_C0060 except no LVA tag

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 99
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization by ETH Zurich 2015

We wanted to know the effect of constitutively expressed aiiA versus no aiiA upon artificial addition of AHL onto Plux.

Genetic design

We designed two plasmids:

  • Medium copy backbone with Plux-GFP and BBa_J23118-BBa_B0032-luxR-BBa_B0012
  • Medium copy backbone with Plux-GFP, BBa_J23118-BBa_B0032-luxR-BBa_B0012 and BBa_J23114-BBa_B0034-aiiA

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (1 µ/mL) and chloramphenicol (0.36 µ/mL) at 37°C and 200 rpm. Cultures were diluted 1:50 in fresh LB with the corresponding antibiotic and transferred to a 96-well plate (200 µL/well). Cultures were grown for 90 min to arrive to exponential phase and then different concentrations of lactate were added. Samples were always made in triplicates and a blank of LB with the corresponding lactate concentration was done. During 7 h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 7 min. The plate was always kept at 37°C. We calculated dose-response curves from the exponential phase of the bacteria.

Modeling

Each experimental data set was fitted to a Hill function using the Least Absolute Residual method (Fitting Toolbox in MatLab). 

    f(AHL) = al+mu*(AHL/K)^n1/(1+(AHL/K)^n1)

Where:

  • al: leakiness in µM/min
  • mu: constant in µM/min
  • Km: half-maximum effective concentration (a representation of sensitivity) in µM
  • n: Hill coefficient (a representation of cooperativity), unitless


PresentAii Absent AiiA (lumped parameters) Absent AiiA (literature parameters)
Coefficient values K=1e+06 (fixed at bound) K=1e+06 (fixed at bound) K = 9.983 (8.929, 11.04)
al = 6134 (5222, 7046) al = 5711 (-2.36e+04, 3.502e+04) al = 2876 (1633, 4119)
mu = 2e+04 (fixed at bound) mu = 2e+05 (fixed at bound) mu = 3.484e+04 (3.338e+04, 3.63e+04)
n1 = 1 (fixed at bound) n1 = 1.646 (-5.203, 8.494) n1 = 1.089 (0.8608, 1.317)
Goodness to fit sse: 5.2051e+09 sse: 3.8192e+12 sse: 1.4366e+09
rsquare: 0.8768 rsquare: -89.3659 rsquare: 0.9986
fe: 10 dfe: 9 dfe: 7
adjrsquare: 0.8768 adjrsquare: -99.4066 adjrsquare: 0.9980
rmse: 2.2815e+04 rmse: 6.5142e+05 rmse: 1.4326e+04
ETH15 NoAiiA.png
ETH15 AiiA.png