Team:Grenoble-EMSE-LSU/Project/Validation

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<h1>Validation</h1>
<h1>Validation</h1>
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<p> We ran 2 experiments proving the stabilization of a bacterial culture, both of them of approximately 14 hours, both of them ddriven by the model. The first one
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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>Experiments</h1>
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<h1>Experiment</h1>
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<h2> </h2>
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<p>In thos experiment we succeed to stabilize the amount of living bacteria at a level of $0.015 OD_{600}$. Even better: the previsions gave us the right amount of bacteria. </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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<br>
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<p>The first part of the experiment followed a protocol close to the other:</p>
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<p>On the curves below:</p>
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<p> <b>1</b>. A first period of 90 minutes in the dark for the bacteria to grow and produce KillerRed. </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> <b>2</b>. A long period of 4 hours at an illumination of maximal intensity. At this time, the effect of illumination were clear, and some parameters could have been adapted to fit these effects.</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 represents experimental datas.</p>
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<br>
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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>
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<img src="https://static.igem.org/mediawiki/2013/2/22/28_1_fluo.png" style="height:300px;width:400px;">
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<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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<br>
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<p> Then the model was used to predict which illumination could stabilized the population.</p>
 
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<p><b>3</b>. Following the model predictions, the illumination of the next period of timehas been at 70% of maximal intensity.</p>
 
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<p><b>4</b>. After 2 hours, as the fluorescence and the absorbance were slowly drifting away from the predictions, the illumination was put at 50% of maximal intensity for 2 hours, and then at 40% of maximal intensity. </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>
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<img src="https://static.igem.org/mediawiki/2013/a/a5/28_2_fluo.png" style="height:300px;width:400px;">
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<img src="https://static.igem.org/mediawiki/2013/e/ee/28_2_OD.png" style="height:300px;width:400px;">
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<TABLE BORDER="1" style="font-size:200%;padding:10px">  
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<p> <b>Step 3:</b></p>
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  <CAPTION> To sum up </CAPTION>  
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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>
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  <TR>  
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<img src="https://static.igem.org/mediawiki/2013/b/b1/28_3_fluo.png" style="height:300px;width:400px;">
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<TH> <b>Time (min)</b> </TH>  
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<img src="https://static.igem.org/mediawiki/2013/5/5a/28_3_OD.png" style="height:300px;width:400px;">
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<TH> 0 </TH>  
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<p> Parameters for these predictions:</p>
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<TH> 90 </TH>  
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<p> $R=83 min$</p>
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<TH> 330 </TH>  
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<p> $a=140 RFU.OD^{-1}.min^{-1}$</p>
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<TH> 450 </TH>  
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<p> $b=0,93.10^{-2}RFU.UL^{-1}.min^{-1}$</p>
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<TH> 570 </TH>  
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<p> $k=0,93.10^{-7}OD.RFU^{-1}.UL^{-1}.min^{-1}$</p>
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<TH> 720 </TH>  
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<p> $l=0.9811$</p>
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  </TR>  
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<p> $M=110 min$</p>
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  <TR>  
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<br>
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<TH> <b>illumination (%)</b> </TH>  
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<TD> 0</TD>  
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<p> <b>Step 4:</b></p>
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<TD> 100 </TD>  
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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>
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<TD> 70 </TD>  
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<img src="https://static.igem.org/mediawiki/2013/c/c5/28_4_fluo.png" style="height:300px;width:400px;">
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<TD> 50 </TD>  
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<img src="https://static.igem.org/mediawiki/2013/8/84/28_4_OD.png" style="height:300px;width:400px;">
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<TD> 40 </TD>  
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<br>
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<TD> end </TD>  
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  </TR>  
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<p> <b>Step 5</b> ($t=570min$):</p>
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</TABLE>  
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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>
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<img src="https://static.igem.org/mediawiki/2013/4/46/28_5_fluo.png" style="height:300px;width:400px;">
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<img src="https://static.igem.org/mediawiki/2013/2/27/28_5_OD.png" style="height:300px;width:400px;">
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<br>
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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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<br>
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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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<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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<br>
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<p>And this is exactly what we obtained (in average).</p>
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<br>
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<p> We have been able to control a bacterial population for 4 consecutive hours .</p>
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<p></p>
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<h2></h2>
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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