Team:Grenoble-EMSE-LSU/Project/Validation

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<h1>Validation</h1>
<h1>Validation</h1>
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<p> To validate our heuristic method, we ran two experiments where we tried to stabilize the concentration of living cells in a bacterial culture during 5 consecutive hours. Both were successful, and we will report in more detail one of them. </p>
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<p> To validate our heuristic method, we ran two experiments where we tried to stabilize the concentration of living cells in a bacterial culture during 4 consecutive hours. Both were successful, and we will report in more detail one of them. </p>
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<h1>Experiment</h1>
<h1>Experiment</h1>
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<p>In the first part of the experiment, we first determined the parameters of the model that describe the behavior of our cell culture, according to the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Density#MCBP">procedure described in the modeling part</a>. </p>
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<p>In the first part of the experiment, we first determined the parameters of the model that describe the behavior of our cell culture, according to the procedure described in the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Density#MCBP">modeling part</a>.</p>
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<p>The first part of the experiment followed a protocol close to the other:</p>
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<p><b>1</b>.0-90 min : this first incubation was done in the dark to let the bacteria grow and produce KillerRed. </p>
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<p><b>2</b>. 90-330 min : a longer 4 hours period with illumination at maximal intensity. After that time, the effects of light were clear, and the parameters of the model were calculated to fit the observable kinetics.</p>
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<p><b>3</b>. 330-450 min : Then our model was used to calculate the light intensity that would stabilize the population of living cells : light was applied at 70% of its maximal intensity.</p>
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<p><b>4</b>. 450-720 min : After 2 hours, we noticed that the fluorescence and the absorbance were slowly drifting away from the predictions. Since the fluorescence was higher than expected and the OD was lower than expected, we reduced the illumination to 50% of its maximal intensity for 2 hours and then at 40% of its maximal intensity. </p>
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<TABLE BORDER="1" style="font-size:180%">
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  <CAPTION> To sum up </CAPTION>
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  <TR >
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<TH style="padding:10px"> <b>Time (min)</b> </TH>
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<TH style="padding:10px"> 0 </TH>
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<TH style="padding:10px"> 90 </TH>
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<TH style="padding:10px"> 330 </TH>
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<TH style="padding:10px"> 450 </TH>
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<TH style="padding:10px"> 570 </TH>
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<TH style="padding:10px"> 720 </TH>
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  </TR>
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  <TR >
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<TH style="padding:10px"> <b>illumination (%)</b> </TH>
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<TD style="padding:10px"> 0</TD>
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<TD style="padding:10px"> 100 </TD>
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<TD style="padding:10px"> 70 </TD>
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<TD style="padding:10px"> 50 </TD>
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<TD style="padding:10px"> 40 </TD>
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<TD style="padding:10px"> end </TD>
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  </TR>
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</TABLE>
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<br>
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<br>
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<p>On the curves below:</p>
<p>On the curves below:</p>
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<p> $\bullet$ The red surface is the prediction of absorbance caused by <b>dead</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($D$, in $OD_{600}$)</a> on the absorbance curve, and the prediction of fluorescence caused by KillerRed stored in <b>dead</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($K_D$, in $UF$)</a> on the fluorescence curve.</p>
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<p> $\bullet$ The red surface is the predicted absorbance of <b>dead</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($D$, in $OD_{600}$)</a> in the absorbance panel, and the predicted KillerRed fluorescence in <b>dead</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($K_D$, in $RFU$)</a> in the fluorescence panel.</p>
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<p> $\bullet$ The yellow surface is the prediction of absorbance caused by <b>living</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($C$, in $OD_{600}$)</a> on the absorbance curve, and the prediction of fluorescence caused by KillerRed stored in <b>living</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($K$, in $UF$)</a> on the fluorescence curve.</p>
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<p> $\bullet$ The yellow surface represents the predicted absorbance of <b>living</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($C$, in $OD_{600}$)</a> in the absorbance panel, and the predicted KillerRedfluorescence KillerRed stored in <b>living</b> bacteria <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building#equation1">($K$, in $RFU$)</a> in the fluorescence panel.</p>
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<p> $\bullet$ The blue line follows the datas collected.</p>
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<p> $\bullet$ The blue line represents experimental datas.</p>
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<p> Step 1: We ran the the experiment to be able to improve the fit of parameters (90 minutes in the dark, then 3 hours at maximal intensity). The predictions below were calculated with parameters of the previous experience, we see that on this one, bacteria are growing slower, and procuce less KillerRed.</p>
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<p> <b>Step 1</b> ($t=330min$):</p>
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<p> This step is divided in two periods : first 1.5 hours in the dark, then 4 hours at maximal intensity of the lamp ($I = 1$). This procedure has 2 objectives : to obtain a precised model and to reach more rapidly the desired density of living bacteria. A first estimate of the cell culture parameters were determined by fitting the curves obtained so far. We then searched for the light profile that would stabilize the cell population. Light intensity is expressed as a fraction or a percentage of the maximum intensity of the bulb. A constant 30% light level could stabilize the living cell density, but it took to much time. We therefore decided to illuminate first at 70% for 2 hours, then to decrease the intensity to 30%. Below are shown the fit of the 5 first hours and the predicted $OD_{600}$ and fluorescence for the upcoming intensities. The cell density should reach its target level (0.02) after 2 hours.</p>
<img src="https://static.igem.org/mediawiki/2013/2/22/28_1_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/2/22/28_1_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/e/e7/28_1_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/e/e7/28_1_OD.png" style="height:300px;width:400px;">
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<p> Parameters for these predictions:</p>
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<p> $R=83 min$</p>
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<p> $a=140 RFU.OD^{-1}.min^{-1}$</p>
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<p> $b=0,9.10^{-2}RFU.UL^{-1}.min^{-1}$</p>
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<p> $k=0,9.10^{-7}OD.RFU^{-1}.UL^{-1}.min^{-1}$</p>
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<p> $l=0.983$</p>
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<p> $M=110 min$</p>
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<p> Step 2: m+330 fit of parameters, new prediction, look for a light tha will stabiliser the bacterial population, here I=0,7.</p>  
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<p> <b>Step 2</b> ($t=450min$):</p>
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<p>After 2 hours, we observe that the fluorescence has closely followed the prediction, but that the absorbance has grown up too fast. The model parameters are thus adjusted again and therefore the predicted kinetics are re-calculated. This may force us to change the light intensity time profile.</p>  
<img src="https://static.igem.org/mediawiki/2013/a/a5/28_2_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/a/a5/28_2_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/e/ee/28_2_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/e/ee/28_2_OD.png" style="height:300px;width:400px;">
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<p> Step 3: m+450 the fluorescence and the OD are too low for the prevision, light intensity is decreased at 0,5.</p>
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<p> <b>Step 3:</b></p>
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<p> Here are the newly predicted kinetics, with the new set of parameters. It seems that an 30% illumination may be insufficient to stabilize the bacterial population.</p>
<img src="https://static.igem.org/mediawiki/2013/b/b1/28_3_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/b/b1/28_3_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/5/5a/28_3_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/5/5a/28_3_OD.png" style="height:300px;width:400px;">
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<p> Parameters for these predictions:</p>
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<p> $R=83 min$</p>
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<p> $a=140 RFU.OD^{-1}.min^{-1}$</p>
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<p> $b=0,93.10^{-2}RFU.UL^{-1}.min^{-1}$</p>
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<p> $k=0,93.10^{-7}OD.RFU^{-1}.UL^{-1}.min^{-1}$</p>
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<p> $l=0.9811$</p>
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<p> $M=110 min$</p>
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<p> Step 4: m+570 the fluorescence and the OD are still too low for the prevision, light intensity is decreased at 0,4.</p>
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<p> <b>Step 4:</b></p>
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<p> The light intensity has to be increased a little bit. Again, to reach the target density more rapidly, we chose a 50% illumination for 2 hours, followed by a 30% illumination. Below are shown the predicted kinetics.</p>
<img src="https://static.igem.org/mediawiki/2013/c/c5/28_4_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/c/c5/28_4_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/8/84/28_4_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/8/84/28_4_OD.png" style="height:300px;width:400px;">
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<p> Step 5: m+660 we have just obtained a level of more than 5 hours</p>
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<p> <b>Step 5</b> ($t=570min$):</p>
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<p> This time, the absorbance fits perfectly the predicted curve, but the fluorescence does not. It instead decreased faster than expected. Just as in 'step 2', the parameters have to be adjusted to improve the fit.</p>
<img src="https://static.igem.org/mediawiki/2013/4/46/28_5_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/4/46/28_5_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/2/27/28_5_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/2/27/28_5_OD.png" style="height:300px;width:400px;">
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<p> <b>Step 6:<b></p>
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<p> Here are the data fitted with the improved set of parameters. Again, the illumination profile needed to be adjusted. 30% illumination was not enough and we increased it to 40%.</p>
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<img src="https://static.igem.org/mediawiki/2013/2/2e/28_6_fluo.png" style="height:300px;width:400px;">
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<img src="https://static.igem.org/mediawiki/2013/1/13/28_6_OD.png" style="height:300px;width:400px;">
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<p> Parameters for these predictions:</p>
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<p> $R=84.5 min$</p>
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<p> $a=140 RFU.OD^{-1}.min^{-1}$</p>
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<p> $b=1.0.10^{-2}RFU.UL^{-1}.min^{-1}$</p>
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<p> $k=0,9.10^{-7}OD.RFU^{-1}.UL^{-1}.min^{-1}$</p>
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<p> $l=0.9807$</p>
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<p> $M=110 min$</p>
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<img src="https://static.igem.org/mediawiki/2013/5/55/Valid_Exp_OD.png" >
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<p> <b>Step 7:<b></p>
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<p> The OD curve is satisfactory, since a 4-hours long linear increase of absorbance can be observed (from $t=360min$ to $t=630min$). This suggests that our procedure works. We still have to compare our results to the number of bacterial colonies able to grow on Petri dishes.</p>
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<img src="https://static.igem.org/mediawiki/2013/3/35/28_7_fluo.png" style="height:300px;width:400px;">
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<img src="https://static.igem.org/mediawiki/2013/f/f9/28_7_OD.png" style="height:300px;width:400px;">
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<img src="https://static.igem.org/mediawiki/2013/8/88/Valid_Exp_fluo.png">
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<p>In the final absorbance curve, the optical density of living cell is about 0.02 ($OD_{600}$). Considering that a cell concentration of $3.10^5 cells/μL$ result in an absorbance of $1 OD_{600}$, we thus expect a colony forming unit concentration of $6000 cells/μL$.</p>
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<img src="https://static.igem.org/mediawiki/2013/d/db/Valid_Nbcolfrancois-2.png">
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<img src="https://static.igem.org/mediawiki/2013/e/e5/Valid_Exp_cells.png">
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<p>And this is exactly what we obtained (in average).</p>
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<img src="https://static.igem.org/mediawiki/2013/d/db/Valid_Nbcolfrancois-2.png">
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<p> We have been able to control a bacterial population for 4 consecutive hours .</p>
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<p>r</p>
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<p></p>
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Latest revision as of 01:25, 5 October 2013

Grenoble-EMSE-LSU, iGEM


Grenoble-EMSE-LSU, iGEM