Difference between revisions of "Team:CCU Taiwan/Model"

Line 191: Line 191:
 
<ul>
 
<ul>
 
<li>
 
<li>
−
<a href="#Fluorescein">Fluorescein curve</a>
+
<a href="#mazef">MazEF modeling</a>
 
<ul>
 
<ul>
−
<li><a href="#Fluorescein-Plate-reader">Plate reader</a></li>
+
<li><a href="#ov">Overview</a></li>
−
<li><a href="#Fluorescein-Material">Material</a></li>
+
<li><a href="#pd">Part description pBAD</a></li>
−
<li><a href="#Fluorescein-Method">Method</a></li>
+
<li><a href="#con">Conclusion</a></li>
−
<li><a href="#Fluorescein-Data-result">Data result</a></li>
+
<li><a href="#ref">Reference</a></li>
 
</ul>
 
</ul>
−
</li>
 
−
<li>
 
−
<a href="#OD600">OD600 Reference point</a>
 
−
      <ul>
 
−
<li><a href="#OD600-Plate-reader">Plate reader</a></li>
 
−
<li><a href="#OD600-Material">Material</a></li>
 
−
<li><a href="#OD600-Method">Method</a></li>
 
−
<li><a href="#OD600-Data-result">Data result</a></li>
 
−
      </ul>
 
−
</li>
 
−
 
−
<li>
 
−
<a href="#Cell">Cell measure</a>
 
−
      <ul>
 
−
<li><a href="#Cell-Material">Material</a></li>
 
−
<li><a href="#Cell-Method">Method</a></li>
 
−
<li><a href="#Cell-Data-result">Data result</a></li>
 
−
      </ul>
 
 
</li>
 
</li>
  
Line 229: Line 211:
  
  
−
<div id="Fluorescein">
+
<div id="mazef">
−
<h2>Fluorescein Fluorescence standard curve</h2>
+
<h2>MazEF modeling</h2>
−
 
+
 
</div>
 
</div>
  
−
<div id="Fluorescein-Plate-reader">
+
<div id="ov">
 
<div class="aaa"></div>
 
<div class="aaa"></div>
−
<h3>Plate reader</h3>
+
<h3>Overview</h3>
 +
<p>
 +
This year modeling plays a great role on our project because we used it to predict the precise time in which our systems start functioning. We showed two essential part modeling below, one is <i>E. coli DH5α</i> and the other one is B. subtilis B. subtilis strain 168(ATCC ® 23857). The showing data will demonstrate our project working result.
 +
</p>
  
−
    <p>
+
<div id="pd">
−
      microplate reader FLUOstar Omega</br>
+
−
emission filter: 520 nm</br>
+
−
excitation filter: 485 nm
+
−
    </p>
+
−
 
+
−
</div>
+
−
 
+
−
<div id="Fluorescein-Material">
+
 
<div class="aaa"></div>
 
<div class="aaa"></div>
−
<h3>Material</h3>
+
<h3>Part description pBAD</h3>
−
<p>
+
−
Fluorescein sodium salt</br>
+
−
1xPBS</br>
+
−
Tissue culture testplate (black with flat bottom)
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
<div id="Fluorescein-Method">
+
−
<div class="aaa"></div>
+
−
                <h3>Method</h3>
+
−
+
−
<ol><li>Prepare fluorescein stock solution</li></ol>
+
 
<p>
 
<p>
−
1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</br>
+
pBAD is the promoter regulated by both arabinose and the araC gene product. Since that araC gene protein regulates expression is also activated by arabinose, pBAD promoter is enormously effected by arabinose. We modeling the relationship between pBAD activity and arabinose concentration for finding out how to let pBAD reach it maximal activity.<br/>
−
2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </br>
+
We assumed that our system reacts as the following chemical system:<br/>
−
3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein  stock solution 50 μM </br></br>
+
 
</p>
 
</p>
−
<ol><li>Serial dilutions</li></ol>
+
<img src="https://static.igem.org/mediawiki/2017/a/a8/Ccum1.png" style="display:block; margin:auto;"><br/>
 +
<img src="https://static.igem.org/mediawiki/2017/f/f8/Ccum2.png" style="display:block; margin:auto;"><br/>
 
<p>
 
<p>
−
1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12</br>
+
Assume that AraC is always in large concentration, the binding reaction between AraC and arabinose is very fast. Thus, we don’t have to consider the concentration of arabinose and AraC. We only need to focus on concentration of Arabinose AraC.
−
2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1</br>
+
To describe the transcription of mRNA, we used Michaelis- Mentin kinetics and get the follow differential equation.
−
3. Transfer 100 μl of fluorescein stock solution from A1 into A2. </br>
+
</p>
−
4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </br>
+
<img src="https://static.igem.org/mediawiki/2017/d/da/Ccum3.png" style="display:block; margin:auto;"><br/>
−
5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </br>
+
<style type="text/css">
−
6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </br>
+
.tg  {border-collapse:collapse;border-spacing:0;}
−
7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </br>
+
.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
−
8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </br>
+
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
−
9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </br>
+
</style>
−
10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </br>
+
<table class="tg">
−
11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</br>  
+
  <tr>
−
12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </br>
+
    <th class="tg-031e">α</th>
−
13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
+
    <th class="tg-031e">Translation rate</th>
−
  (Caution: Do not to continue serial dilution into column 12.)</br>
+
    <th class="tg-031e">15ntds−1/length of sequence</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">γ1</td>
 +
    <td class="tg-031e">Combineddegradation and dilution rate,of mRNA</td>
 +
    <td class="tg-031e">2.2×10-3(S-1)</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">γ2</td>
 +
    <td class="tg-031e">Combineddegradation and,dilution rate of GFP</td>
 +
    <td class="tg-031e">5.2×10-4(S-1)</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">Kmax</td>
 +
    <td class="tg-031e">Maximal transcription rate</td>
 +
    <td class="tg-031e">50ntd,S-1/length of sequence</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">Khalf</td>
 +
    <td class="tg-031e">Half-maximal transcription,rate</td>
 +
    <td class="tg-031e">160μM</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">n</td>
 +
    <td class="tg-031e">Hill coefficient</td>
 +
    <td class="tg-031e">2.65</td>
 +
  </tr>
 +
</table>
 +
<p>The above data is all from 2015 Oxford iGEM modeling<br/><br/>
 +
Using Polymath, we can get the different signal of GFP in different concentration of Arabinose-AraC.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2017/4/46/Ccum5.png" style="display:block; margin:auto;"><br/>
 +
<p>Figure 1 The respond of GFP in different concentration of Arabinose-AraC. Range 0.13 μM~10μM<br/>
 +
We can see that different concentration of Arabinose-AraC will affect the maximum amount of GFP production. We also can see that they reach maximum signal at the same time at approximate 60 minute.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2017/c/c2/Ccum6.png" style="display:block; margin:auto;"><br/>
 +
<p>Figure 2 The respond of GFP in different concentration of Arabinose-AraC. Range 6000 μM~130000μM<br/>
 +
In high concentration of arabinose, we can see that all the line will overlap. The signal of GFP isn’t changing.
 
</p>
 
</p>
−
<ol><li>repeat serial dilute for Row B、D、E</strong></li></ol>
 
−
<ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol>
 
−
<ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol>
 
−
 
−
<br/><br/>
 
−
 
 
</div>
 
</div>
  
−
<div id="Fluorescein-Data-result">
+
<div id="con">
 
<div class="aaa"></div>
 
<div class="aaa"></div>
−
<h3>Data result</h3>
+
<h3>Conclusion:</h3>
−
<br/>
+
−
<img src="https://static.igem.org/mediawiki/2017/8/85/FFs_1.png" style="display:block; margin:auto;"><br/><br/>
+
−
<img src="https://static.igem.org/mediawiki/2017/a/a4/FFs_2.png" style="display:block; margin:auto;"><br/><br/>
+
−
<img src="https://static.igem.org/mediawiki/2017/5/5c/FFs_3.png" style="display:block; margin:auto;"><br/>
+
−
</div>
+
−
</section>
+
−
 
+
−
<section>
+
−
 
+
−
 
+
−
<div id="OD600">
+
−
<h2>OD600 Reference point</h2>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div id="OD600-Plate-reader">
+
−
<div class="aaa"></div>
+
−
<h3>Plate reader</h3>
+
−
 
+
−
    <p>
+
−
Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</br>
+
−
Filter: 595 nm</br>
+
−
    </p>
+
−
 
+
−
</div>
+
−
 
+
−
<div id="OD600-Material">
+
−
  <div class="aaa"></div>
+
−
<h3>Material</h3>
+
−
<p>
+
−
1 ml LUDOX</br>
+
−
mQH<sub>2</sub>O</br>
+
−
96 well cell culture plate (clear with flat-bottom)
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
<div id="OD600-Method">
+
−
<div class="aaa"></div>
+
−
                <h3>Method</h3>
+
−
 
+
 
<p>
 
<p>
−
1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</br>
+
Assume that we want to make a difference in our system, the amount of arabinose shouldn’t be too large, and the interval should be too large either. Because pBAD system is very sensitive to arabinose, a trivial change can result in drastic impact. And we found out that the system would reach equilibrium at approximately 60 mins. So we learned that the kill switch would be activated after 1 hour. So the sampling point interval can be roughly 1 hour.
−
2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</br>
+
−
3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</br>
+
−
4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</br>
+
 
</p>
 
</p>
−
 
 
</div>
 
</div>
  
−
<div id="OD600-Data-result">
+
<div id="ref">
−
<div class="aaa"></div>
+
−
<h3>Data result</h3>
+
−
<br/>
+
−
<img src="https://static.igem.org/mediawiki/2017/c/cd/Ee.jpeg" style="display:block; margin:auto;"><br/>
+
−
</div>
+
−
</section>
+
−
 
+
−
<section>
+
−
 
+
−
 
+
−
<div id="Cell">
+
−
<h2>Cell measure</h2>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div id="Cell-Material">
+
−
  <div class="aaa"></div>
+
−
<h3>Material</h3>
+
−
<p>
+
−
Competent cells ( Escherichia coli strain DH5α)</br>
+
−
LB (Luria Bertani) media</br>
+
−
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</br>
+
−
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</br>
+
−
Incubator at 37°C</br>
+
−
1.5 ml eppendorf tubes for sample storage</br>
+
−
Ice bucket with ice</br>
+
−
Pipettes</br>
+
−
96 well plate(cell culture 96 well plate、tissue culture testplate)</br>
+
−
Devices (from InterLab Measurement Kit):</br>
+
−
1. Negative control(BBa_R0040)</br>
+
−
2. Positive control(J23151+B0032+E0040+B0010+B0012)</br>
+
−
3. Test Device 1: J23101+I13504</br>
+
−
4. Test Device 2: J23106+I13504</br>
+
−
5. Test Device 3: J23117+I13504</br>
+
−
6. Test Device 4: J23101+BCD2+E0040+B0015</br>
+
−
7. Test Device 5: J23106+BCD2+E0040+B0015</br>
+
−
8. Test Device 6: J23117+BCD2+E0040+B0015</br>
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
<div id="Cell-Method">
+
 
<div class="aaa"></div>
 
<div class="aaa"></div>
−
                <h3>Method</h3>
+
<h3>Reference:</h3>
−
 
+
 
<p>
 
<p>
−
1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</br>
+
1. Ben-Samoun, K., Leblon, G., & Reyes, O. (1999). Positively regulated expression of the Escherichia coli araBAD promoter in Corynebacterium glutamicum. FEMS microbiology letters, 174(1), 125-130. <br/>
−
&nbsp;(Transformation protocol is from iGEM)</br>
+
2. Guzman, L.-M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121-4130. <br/>
−
2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</br>
+
3. https://2015.igem.org/Team:Oxford/Modeling
−
3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</br>
+
−
4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</br>
+
−
5. Incubate the cultures at 37°C and 170 rpm.</br>
+
−
6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</br>
+
−
7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</br>
+
 
</p>
 
</p>
  
−
</div>
 
−
 
−
<div id="Cell-Data-result">
 
−
<div class="aaa"></div>
 
−
<h3>Data result</h3>
 
−
<br/>
 
−
<img src="https://static.igem.org/mediawiki/2017/f/f7/IL_cell1.jpg" style="display:block; margin:auto;"><br/><br/>
 
−
<img src="https://static.igem.org/mediawiki/2017/4/41/IL_cell2.jpg" style="display:block; margin:auto;"><br/>
 
−
</div>
 
 
</section>
 
</section>
  

Revision as of 03:52, 2 November 2017

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MazEF modeling

Overview

This year modeling plays a great role on our project because we used it to predict the precise time in which our systems start functioning. We showed two essential part modeling below, one is E. coli DH5α and the other one is B. subtilis B. subtilis strain 168(ATCC ® 23857). The showing data will demonstrate our project working result.

Part description pBAD

pBAD is the promoter regulated by both arabinose and the araC gene product. Since that araC gene protein regulates expression is also activated by arabinose, pBAD promoter is enormously effected by arabinose. We modeling the relationship between pBAD activity and arabinose concentration for finding out how to let pBAD reach it maximal activity.
We assumed that our system reacts as the following chemical system:



Assume that AraC is always in large concentration, the binding reaction between AraC and arabinose is very fast. Thus, we don’t have to consider the concentration of arabinose and AraC. We only need to focus on concentration of Arabinose AraC. To describe the transcription of mRNA, we used Michaelis- Mentin kinetics and get the follow differential equation.


α Translation rate 15ntds−1/length of sequence
γ1 Combineddegradation and dilution rate,of mRNA 2.2×10-3(S-1)
γ2 Combineddegradation and,dilution rate of GFP 5.2×10-4(S-1)
Kmax Maximal transcription rate 50ntd,S-1/length of sequence
Khalf Half-maximal transcription,rate 160μM
n Hill coefficient 2.65

The above data is all from 2015 Oxford iGEM modeling

Using Polymath, we can get the different signal of GFP in different concentration of Arabinose-AraC.


Figure 1 The respond of GFP in different concentration of Arabinose-AraC. Range 0.13 μM~10μM
We can see that different concentration of Arabinose-AraC will affect the maximum amount of GFP production. We also can see that they reach maximum signal at the same time at approximate 60 minute.


Figure 2 The respond of GFP in different concentration of Arabinose-AraC. Range 6000 μM~130000μM
In high concentration of arabinose, we can see that all the line will overlap. The signal of GFP isn’t changing.

Conclusion:

Assume that we want to make a difference in our system, the amount of arabinose shouldn’t be too large, and the interval should be too large either. Because pBAD system is very sensitive to arabinose, a trivial change can result in drastic impact. And we found out that the system would reach equilibrium at approximately 60 mins. So we learned that the kill switch would be activated after 1 hour. So the sampling point interval can be roughly 1 hour.

Reference:

1. Ben-Samoun, K., Leblon, G., & Reyes, O. (1999). Positively regulated expression of the Escherichia coli araBAD promoter in Corynebacterium glutamicum. FEMS microbiology letters, 174(1), 125-130.
2. Guzman, L.-M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121-4130.
3. https://2015.igem.org/Team:Oxford/Modeling