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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<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 collected datas.</p>
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<p> $\bullet$ The blue line represents experimental datas.</p>
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<p> <b>Step 1</b> ($t=300min$):</p>
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<p> <b>Step 1</b> ($t=330min$):</p>
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<p> The experiment is run for 5 hours without control. There is first a period of 1 hour and a half in the dark, then 3 hours and a half at maximal intensity. This process has 2 objectives : to fasten the emergence of the level of the amount of living bacteria and to improve the precision of model. At this point, parameters are found to fit best the curves observed, that is the reason why a long period is needed : it will improve the precision of parameters. Then, we search for the light the will stabilize the population cell. It could have been 30% of maximal intensity all along, but again to fasten the apparition of the level, we decide to illuminate bacteria first at 70% for 2 hours, and then decrease the intensity at 30%. Below are the fit of the 5 first hours and the predictions for the chosen intensities. The level should appear in 2 hours.</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> <b>Step 2</b> ($t=450min$):</p>
<p> <b>Step 2</b> ($t=450min$):</p>
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<p>After 2 hours, we observes that the fluorescence has followed the prediction, but the absorbance has grown up too fast. Parameters have to be changed and therefore new predictions have to be calculated. At the end, we may have to change the plan of light intensity.</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> <b>Step 3:</b></p>
<p> <b>Step 3:</b></p>
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<p> Here are the new prediction, with new parameters. Unfortunately, it seems that an illumination at 30% is a little too weak to stabilize the population.</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> <b>Step 4:</b></p>
<p> <b>Step 4:</b></p>
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<p> The light intensity has to be increase a little bit. Again, to accelerate the emergence of the level, we chose an illumination of 50% for 2 hours, and then an illumination of 30%. Below are the prediction with this illumination.</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> <b>Step 5</b> ($t=570min$):</p>
<p> <b>Step 5</b> ($t=570min$):</p>
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<p> This time, the absorbance has been perfectly following the predictions, but the fluorescence has not, it has decreased faster than what was predicted. Just like in 'step 2', the parameters have to be changed to improve the fit.</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>
<p> <b>Step 6:<b></p>
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<p> Here are the new predictions. We even have concentrated 2 steps in these kinetics for the future illumination has been changed. For this prediction, 30% of maximal illumination was not enough to stabilize the bacterial population, therefore it has been increase to 40%.</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>
<img src="https://static.igem.org/mediawiki/2013/2/2e/28_6_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/2/2e/28_6_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/1/13/28_6_OD.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/1/13/28_6_OD.png" style="height:300px;width:400px;">
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<p> <b>Step 7:<b></p>
<p> <b>Step 7:<b></p>
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<p> Here are the new predictions. We even have concentrated 2 steps in these kinetics for the future illumination has been changed. For this prediction, 30% of maximal illumination was not enough to stabilize the bacterial population, therefore it has been increase to 40%.</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>
<img src="https://static.igem.org/mediawiki/2013/3/35/28_7_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/3/35/28_7_fluo.png" style="height:300px;width:400px;">
<img src="https://static.igem.org/mediawiki/2013/f/f9/28_7_OD.png" style="height:300px;width:400px;">
<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/d/db/Valid_Nbcolfrancois-2.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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<p> The experiment has been continued to create a new set of parameters. Here are the new predictions, with the parameters value of the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Parameters">fifth experiment</a>.</p>
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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/2013/5/55/Valid_Exp_OD.png" >
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<p> We have been able to control a bacterial population for 4 consecutive hours .</p>
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<img src="https://static.igem.org/mediawiki/2013/8/88/Valid_Exp_fluo.png">
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<img src="https://static.igem.org/mediawiki/2013/e/e5/Valid_Exp_cells.png">
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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