Difference between revisions of "Team:Ionis Paris/Protocol 13"

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                               <h4 class="blog" style="margin-top:20px">
 
                               <h4 class="blog" style="margin-top:20px">
 
                                     Toluene preparation: stock solutions</h4>
 
                                     Toluene preparation: stock solutions</h4>
−
                                    <p><b><u>100mg/L:</u></b> Dilution of absolute liquid toluene to obtain a final toluene concentration at 100mg/L: Add 11,53µL of toluene into 100mL water (calculations done from toluene's molecular weight)<br/><b><u>100µg/L:</u></b> Dilute 10µL of the first stock solution into 10mL of water</p>
+
 
 +
<p>
 +
<b><u>1g/L:</u></b> Dilution of absolute liquid toluene to obtain a final toluene concentration at 1g/L: Add 11,53µL of toluene into 10mL water (calculations done from toluene's molecular weight) <br/>
 +
 
 +
<b><u>100mg/L:</u></b> Dilute 1mL of the toluene stock solution at 1g/L into 9mL of water<br/>
 +
 
 +
<b><u>100µg/L:</u></b> Dilute 10µL of the toluene stock solution at 100mg/L into 10mL of water</p>
  
 
                               <h4 class="blog" style="margin-top:20px">
 
                               <h4 class="blog" style="margin-top:20px">
 
                                     Inoculation</h4>
 
                                     Inoculation</h4>
−
                                 <p>From 1 UFC:<br./>
+
                                 <p>From 1 UFC:<br/>
−
Resuspend colony in 20mL of LB medium containing chloramphenicol at 25µg/mL but free of toluene and grow over night.<br/>
+
Resuspend a colony in 20mL of LB medium containing chloramphenicol at 25µg/mL but free of toluene and grow over night.<br/>
 
When turbidity is high, inoculate the appropriate volume of bacteria culture to liquid LB medium containing chloramphenicol to reach 0,1 OD in the flask (100mL). Incubate culture at 37°C and 250 rpm .<br/>
 
When turbidity is high, inoculate the appropriate volume of bacteria culture to liquid LB medium containing chloramphenicol to reach 0,1 OD in the flask (100mL). Incubate culture at 37°C and 250 rpm .<br/>
 
When the OD reach 0.3, separate the initial bacterial culture into several falcon tubes (10mL in each tube) and add toluene to reach the desired toluene concentration. </p>
 
When the OD reach 0.3, separate the initial bacterial culture into several falcon tubes (10mL in each tube) and add toluene to reach the desired toluene concentration. </p>
  
 
<table>
 
<table>
 +
 
<tr>
 
<tr>
 +
<td> </td>
 +
<td> C0 Control </td>
 +
<td> C1 </td>
 +
<td> C2 </td>
 +
</tr>
  
 +
<tr>
 +
<td>  Bacterial culture (mL) </td>
 +
<td> 10 </td>
 +
<td> 10 </td>
 +
<td> 10 </td>
 
</tr>
 
</tr>
  
 +
<tr>
 +
<td> Toluene from stock solution 100µg/L (µL) </td>
 +
<td> 0 </td>
 +
<td> 1 </1td>
 +
<td> 10 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Obtain concentration </td>
 +
<td> 0 </td>
 +
<td> 10ng/L </td>
 +
<td> 100ng/L </td>
 +
</tr>
 
</table>
 
</table>
  
 +
<table>
 +
<tr>
 +
<td> </td>
 +
<td> C3 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Bacterial culture  (mL) </td>
 +
<td> 10 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Toluene from stock solution 100mg/L (µL)  </td>
 +
<td> 1 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Obtain concentration </td>
 +
<td> 10µg/L </td>
 +
</tr>
 +
</table>
 +
 +
<table>
 +
<tr>
 +
<td> </td>
 +
<td> C4 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Bacterial culture (mL) </td>
 +
<td> 10 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Toluene from stock solution 1g/L (µL) </td>
 +
<td> 100 </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Obtain concentration </td>
 +
<td> 10mg/L </td>
 +
</tr>
 +
</table>
  
  
  
 
                                 <p>Incubate culture at 37°C and 250 rpm.<br/>
 
                                 <p>Incubate culture at 37°C and 250 rpm.<br/>
−
Prepare the GLuc assay solution (e.g. 100 samples) by adding 50 μl of BioLux GLuc Substrate to 5 ml of BioLux GLuc Assay Buffer immediately before performing the assay. Mix well by inverting the tube several times (Do not vortex). Set the camera.<br/>
+
Prepare the GLuc assay solution (e.g. 100 samples) by adding 50µL of BioLux GLuc Substrate to 5mL of BioLux GLuc Assay Buffer immediately before performing the assay. Mix well by inverting the tube several times (Do not vortex). Set the camera.<br/>
−
At several time (t= 30min, t=1h, t=1h30…, t=4h), pipet samples (5–20 μl per well) into a 96-well white (opaque) or black plate, or a luminometer tube. <br/>
+
At several time (t=1h, t=3h…,t=4h30, t=5h30), pipet samples (20µL per well) into a 96-well white (opaque) or black plate, or a luminometer tube. <br/>
−
Add the GLuc assay solution (50 μl) to a sample (i.e. Add the assay solution to only one sample at a time) and promptly measure the luminescence. <br/>
+
Add 50µL of GLuc assay solution to a sample (i.e. Add the assay solution to only one sample at a time) and promptly measure the luminescence. <br/>
−
<b>NB : Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate. </b></p>
+
<i>NB : Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate. </i></p>
  
 
<iframe width="560" height="315" src="https://www.youtube.com/embed/G0SF4aWuL4E" frameborder="0" allowfullscreen></iframe>
 
<iframe width="560" height="315" src="https://www.youtube.com/embed/G0SF4aWuL4E" frameborder="0" allowfullscreen></iframe>

Revision as of 06:57, 17 October 2016

Protocol 13: Luciferase assay

Aim: Assess the luminescence of our biosensor when exposed to toluene

3 replicates for each toluene concentration were realized in order to improve the results statistics

Toluene preparation: stock solutions

1g/L: Dilution of absolute liquid toluene to obtain a final toluene concentration at 1g/L: Add 11,53µL of toluene into 10mL water (calculations done from toluene's molecular weight)
100mg/L: Dilute 1mL of the toluene stock solution at 1g/L into 9mL of water
100µg/L: Dilute 10µL of the toluene stock solution at 100mg/L into 10mL of water

Inoculation

From 1 UFC:
Resuspend a colony in 20mL of LB medium containing chloramphenicol at 25µg/mL but free of toluene and grow over night.
When turbidity is high, inoculate the appropriate volume of bacteria culture to liquid LB medium containing chloramphenicol to reach 0,1 OD in the flask (100mL). Incubate culture at 37°C and 250 rpm .
When the OD reach 0.3, separate the initial bacterial culture into several falcon tubes (10mL in each tube) and add toluene to reach the desired toluene concentration.

C0 Control C1 C2
Bacterial culture (mL) 10 10 10
Toluene from stock solution 100µg/L (µL) 0 1 10
Obtain concentration 0 10ng/L 100ng/L
C3
Bacterial culture (mL) 10
Toluene from stock solution 100mg/L (µL) 1
Obtain concentration 10µg/L
C4
Bacterial culture (mL) 10
Toluene from stock solution 1g/L (µL) 100
Obtain concentration 10mg/L

Incubate culture at 37°C and 250 rpm.
Prepare the GLuc assay solution (e.g. 100 samples) by adding 50µL of BioLux GLuc Substrate to 5mL of BioLux GLuc Assay Buffer immediately before performing the assay. Mix well by inverting the tube several times (Do not vortex). Set the camera.
At several time (t=1h, t=3h…,t=4h30, t=5h30), pipet samples (20µL per well) into a 96-well white (opaque) or black plate, or a luminometer tube.
Add 50µL of GLuc assay solution to a sample (i.e. Add the assay solution to only one sample at a time) and promptly measure the luminescence.
NB : Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate.