Difference between revisions of "Team:Toulouse/Experiments"

Line 233: Line 233:
 
<li>Pump wich expels air</li>
 
<li>Pump wich expels air</li>
 
<li>15 mL Flacon tube</li>
 
<li>15 mL Flacon tube</li>
−
<li>Plastic pipe <br> Ø = 10mm</li>
+
<li>Plastic pipe <br> Ø = 10 mm</li>
−
<li>Glass T pipe <br> Ø = 10mm, made by a glassworker</li>
+
<li>Glass T pipe <br> Ø = 10 mm, made by a glassworker</li>
 
<li>Plastic separator</li>
 
<li>Plastic separator</li>
 
<li>Carded cotton</li>
 
<li>Carded cotton</li>
Line 271: Line 271:
 
our thoughts on present treatments. When beekeepers use formic acid for long  
 
our thoughts on present treatments. When beekeepers use formic acid for long  
 
treatment they place a diffuser at the top of the hive and formic acid concentration  
 
treatment they place a diffuser at the top of the hive and formic acid concentration  
−
was assessed at 200ppm<a target="_blank" href="http://www.agroscope.admin.ch/imkerei/00316/00329/02079/index.html?lang=en"> <SUP>1</SUP> </a>on average that is equivalent to 7,826 mmol.m<SUP>-3</SUP>.  
+
was assessed at 200ppm<a target="_blank" href="http://www.agroscope.admin.ch/imkerei/00316/00329/02079/index.html?lang=en"> <SUP>1</SUP> </a>on average that is equivalent to 7.826 mmol.m<SUP>-3</SUP>.  
 
As gas concentration is difficult to evaluate we calculate the liquid concentration balance   
 
As gas concentration is difficult to evaluate we calculate the liquid concentration balance   
 
thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.
 
thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.
Line 282: Line 282:
 
$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$
 
$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$
 
<!-- P. V = n.R.T, ideal gaz law-->
 
<!-- P. V = n.R.T, ideal gaz law-->
−
$$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$
+
$$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8.314\times293=19,964 Pa $$
 
</p>
 
</p>
 
</div>
 
</div>
Line 288: Line 288:
 
<li>P<SUB>A</SUB>: partial pressure of A in Pa</li>
 
<li>P<SUB>A</SUB>: partial pressure of A in Pa</li>
 
<li>C<SUB>A</SUB>: Concentration of A in air in mol.m<SUP>-3</SUP></li>
 
<li>C<SUB>A</SUB>: Concentration of A in air in mol.m<SUP>-3</SUP></li>
−
<li>R: perfect gaz constant = 8,314 J.mol<SUP>-1</SUP>.K<SUP>-1</SUP></li>
+
<li>R: perfect gaz constant = 8.314 J.mol<SUP>-1</SUP>.K<SUP>-1</SUP></li>
 
<li>T: temperature in <SUP>°</SUP>K </li>
 
<li>T: temperature in <SUP>°</SUP>K </li>
 
</ul>
 
</ul>
Line 294: Line 294:
 
<div class="group center" style="padding-top:10px;">
 
<div class="group center" style="padding-top:10px;">
 
<p style="font-size:15px;">
 
<p style="font-size:15px;">
−
<!-- P_A = C_A.R.T = 7,826.10^3 x 8,314 x 293 = 19,964 Pa -->
+
<!-- P_A = C_A.R.T = 7,826.10^3 x 8.314 x 293 = 19,964 Pa -->
 
$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$
 
$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$
 
<!-- P_A = H_A . C_{A,eq}, Henry's law -->
 
<!-- P_A = H_A . C_{A,eq}, Henry's law -->
Line 371: Line 371:
 
<div class="group">
 
<div class="group">
 
<ul style="font-size:15px;"align="left">
 
<ul style="font-size:15px;"align="left">
−
<li>Butyric acid : 218mM, 109mM, 10.9mM, 5.45mM and 1.09mM</li>
+
<li>Butyric acid : 218 mM, 109 mM, 10.9 mM, 5.45 mM and 1.09 mM</li>
−
<li>Formic acid : 100mM, 10mM, 1mM 500µM, 100µM, 50µM and 25µM </li>
+
<li>Formic acid : 100 mM, 10 mM, 1 mM 500 µM, 100 µM, 50 µM and 25 µM </li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 386: Line 386:
 
<li>Pre-culture of <i>E. coli</i> BW 25113</li>
 
<li>Pre-culture of <i>E. coli</i> BW 25113</li>
 
<li>Acid solutions</li>
 
<li>Acid solutions</li>
−
<li>Medium : LB, M9 15mM of glucose or 30mM of glucose</li>
+
<li>Medium : LB, M9 15 mM of glucose or 30 mM of glucose</li>
 
</div>
 
</div>
  
Line 394: Line 394:
 
</div>
 
</div>
 
<ol align="justify" style="font-size:15px;">
 
<ol align="justify" style="font-size:15px;">
−
<li>Add 400µL of medium in each well</li>
+
<li>Add 400 µL of medium in each well</li>
−
<li>Add 50µL of pre-culture</li>
+
<li>Add 50 µL of pre-culture</li>
−
<li>Add 50µL of acid solution</li>
+
<li>Add 50 µL of acid solution</li>
 
<li>Place the 48 well plate in the optical reader</li>
 
<li>Place the 48 well plate in the optical reader</li>
 
<li>Adjust parameters on computer. <br>  
 
<li>Adjust parameters on computer. <br>  
Line 428: Line 428:
 
<li>Erlenmeyers</li>
 
<li>Erlenmeyers</li>
 
<li>TubeSpin<SUP>®</SUP> Bioreactors from TPP brand</li>
 
<li>TubeSpin<SUP>®</SUP> Bioreactors from TPP brand</li>
−
<li>Medium : M9 15mM of glucose or 30mM of glucose</li>
+
<li>Medium : M9 15 mM of glucose or 30 mM of glucose</li>
 
<li>Incubators at 37°C, 130rpm and without agitation</li>
 
<li>Incubators at 37°C, 130rpm and without agitation</li>
 
<li>1.5mL Eppendorf</li>
 
<li>1.5mL Eppendorf</li>
Line 439: Line 439:
 
</div>
 
</div>
 
<ol align="justify" style="font-size:15px;">
 
<ol align="justify" style="font-size:15px;">
−
<li>Add 50mL of medium on erlenmyer and TubeSpin<SUP>®</SUP> Bioreactor</li>
+
<li>Add 50 mL of medium on erlenmyer and TubeSpin<SUP>®</SUP> Bioreactor</li>
 
<li>Inoculate from pre-culture to have OD<SUB>600nm</SUB>=0.1. <br>
 
<li>Inoculate from pre-culture to have OD<SUB>600nm</SUB>=0.1. <br>
 
To do that centrifuge the appropriate volume of pre-culture, then remove LB medium and resuspend sediment with M9 medium to inoculate.<br>
 
To do that centrifuge the appropriate volume of pre-culture, then remove LB medium and resuspend sediment with M9 medium to inoculate.<br>
Line 446: Line 446:
 
<li>Sampling every two hours the first day:</li>
 
<li>Sampling every two hours the first day:</li>
 
<ul>
 
<ul>
−
<li>Take 1mL of culture in 1.5mL Eppendorf. <br>
+
<li>Take 1mL of culture in 1.5 mL Eppendorf. <br>
 
Attention: for TubeSpin<SUP>®</SUP> Bioreactor use needle and syringe in order not to let air enter, as possible.
 
Attention: for TubeSpin<SUP>®</SUP> Bioreactor use needle and syringe in order not to let air enter, as possible.
 
</li>
 
</li>
−
<li>Add 100µL of sample in spectrophotometer cuvette, complete with 900µL water and measure OD<SUB>600nm</SUB> with spectrophotometer</li>
+
<li>Add 100 µL of sample in spectrophotometer cuvette, complete with 900µL water and measure OD<SUB>600nm</SUB> with spectrophotometer</li>
−
<li>Centrifuge the rest of samples at 13000rpm during 3 minutes</li>
+
<li>Centrifuge the rest of samples at 13000 rpm during 3 minutes</li>
−
<li>Filter the supernatant through a 0.2µm filter and conserve it at -20°C
+
<li>Filter the supernatant through a 0.2 µm filter and conserve it at -20°C
 
</li>
 
</li>
 
</ul>
 
</ul>
Line 474: Line 474:
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
 
<li>Culture supernatants from -20°C</li>
 
<li>Culture supernatants from -20°C</li>
−
<li>2.5mM TSP (Trimethylsilyl propanoic acid) diluted in D<SUB>2</SUB>O
+
<li>2.5 mM TSP (Trimethylsilyl propanoic acid) diluted in D<SUB>2</SUB>O
 
</li>
 
</li>
−
<li>0.5mm NMR tubes</li>
+
<li>0.5 mm NMR tubes</li>
 
<li>1.5mL Eppendorf</li>
 
<li>1.5mL Eppendorf</li>
 
<li>Spinners (5mm)</li>
 
<li>Spinners (5mm)</li>
Line 487: Line 487:
 
</div>
 
</div>
 
<ol align="justify" style="font-size:15px;">
 
<ol align="justify" style="font-size:15px;">
−
<li>Add 400µL of culture supernatant in 1.5mL Eppendorf
+
<li>Add 400 µL of culture supernatant in 1.5mL Eppendorf
 
</li>
 
</li>
−
<li>Add 100µL of TSP solution</li>
+
<li>Add 100 µL of TSP solution</li>
−
<li>Place the mix in 0.5mm NMR tubes</li>
+
<li>Place the mix in 0.5 mm NMR tubes</li>
 
<li>Place NMR tube into spinner, sample is ready to analyse</li>
 
<li>Place NMR tube into spinner, sample is ready to analyse</li>
 
 
Line 580: Line 580:
 
</div>
 
</div>
 
<ol align="justify" style="font-size:15px;">
 
<ol align="justify" style="font-size:15px;">
−
<li>For each standard solutions : in a 1.5mL Eppendorf sample 10µL and add 1mL Bradford’s Reagent, wait 20 minutes and measure OD<SUB>505nm</SUB>
+
<li>For each standard solutions : in a 1.5 mL Eppendorf sample 10 µL and add 1mL Bradford’s Reagent, wait 20 minutes and measure OD<SUB>505nm</SUB>
 
</li>
 
</li>
 
<li>Plot glucose concentration in function of OD<SUB>505nm</SUB> and determine the linear region
 
<li>Plot glucose concentration in function of OD<SUB>505nm</SUB> and determine the linear region
 
</li>
 
</li>
−
<li>Add 1.5mL of BioSilta medium and 45µL of reagent A (50U/L) in an Eppendorf
+
<li>Add 1.5 mL of BioSilta medium and 45 µL of reagent A (50 U/L) in an Eppendorf
 
</li>
 
</li>
 
<li>Sampling every 30 minutes: </li>
 
<li>Sampling every 30 minutes: </li>
 
<ol>
 
<ol>
−
<li>Take 10µL and add 1mL of Bradford’s reagent in an Eppendorf
+
<li>Take 10 µL and add 1 mL of Bradford’s reagent in an Eppendorf
 
</li>
 
</li>
 
<li>Wait 20 minutes
 
<li>Wait 20 minutes
Line 649: Line 649:
 
</div>
 
</div>
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li>4h culture in 50mL Falcon in M9 medium with 15mM of glucose</li>
+
<li>4 h culture in 50 mL Falcon in M9 medium with 15 mM of glucose</li>
 
<li>Silicon plugs adapted to 50mL Falcon</li>
 
<li>Silicon plugs adapted to 50mL Falcon</li>
 
<li>Needles
 
<li>Needles
 
</li>
 
</li>
−
<li>0.2µm filters</li>
+
<li>0.2 µm filters</li>
−
<li>10mL Syringes
+
<li>10 mL Syringes
 
</li>
 
</li>
−
<li>Neoprene pipes Ø=0.8mm
+
<li>Neoprene pipes Ø=0.8 mm
 
</li>
 
</li>
−
<li>10mM NaHCO<SUB>3</SUB></li>
+
<li>10 mM NaHCO<SUB>3</SUB></li>
 
<li>1.5mL Eppendorf</li>
 
<li>1.5mL Eppendorf</li>
 
<li>1mL Sterile cone</li>
 
<li>1mL Sterile cone</li>
Line 686: Line 686:
 
<p style="font-size:15px;" align="justify">
 
<p style="font-size:15px;" align="justify">
 
<u>Remark 1:</u> We used culture in M9 because with the “Acids production tests” we had data on this medium.<br>
 
<u>Remark 1:</u> We used culture in M9 because with the “Acids production tests” we had data on this medium.<br>
−
<u>Remark 2:</u> 10mM NaHCO<SUB>3</SUB> solution was used because pH was 8.3 so it would permit acid gas solubilisation.
+
<u>Remark 2:</u> 10 mM NaHCO<SUB>3</SUB> solution was used because pH was 8.3 so it would permit acid gas solubilisation.
 
</p>
 
</p>
 
</div>  
 
</div>  
Line 712: Line 712:
 
<li>TPX, gas permeable plastic</li>
 
<li>TPX, gas permeable plastic</li>
 
<li>Fusing machine</li>
 
<li>Fusing machine</li>
−
<li>2mM Formic acid solution</li>
+
<li>2 mM Formic acid solution</li>
−
<li>4% (V/V) Butyric acid solution</li>
+
<li>4 % (V/V) Butyric acid solution</li>
 
</div>
 
</div>
  
Line 760: Line 760:
 
         <thead>
 
         <thead>
 
           <tr>
 
           <tr>
−
             <th>YETM 500mL</th>
+
             <th>YETM 500 mL</th>
−
             <th>TFB1 200mL</th>
+
             <th>TFB1 200 mL</th>
−
             <th>TFB2 200mL</th>
+
             <th>TFB2 200 mL</th>
 
           </tr>
 
           </tr>
 
         </thead>
 
         </thead>
Line 769: Line 769:
 
             <td><!-- YETM 500mL -->
 
             <td><!-- YETM 500mL -->
 
<ul style="font-size:15px;"align="left">
 
<ul style="font-size:15px;"align="left">
−
<li> 2.5g Yeast Extract </li>
+
<li> 2.5 g Yeast Extract </li>
−
<li> 10g Tryptone </li>
+
<li> 10 g Tryptone </li>
−
<li> 5g MgSO4.7H<SUB>2</SUB>O </li>
+
<li> 5 g MgSO4.7H<SUB>2</SUB>O </li>
 
<li> Adjust pH to 7.5 with KOH </li>
 
<li> Adjust pH to 7.5 with KOH </li>
−
<li> <b>For Plates</b>: add 7.5g of Agar </li>
+
<li> <b>For Plates</b>: add 7.5 g of Agar </li>
 
</ul>
 
</ul>
 
</td><!-- YETM 500mL END -->
 
</td><!-- YETM 500mL END -->
 
 
−
<td><!-- TFB1 200mL -->
+
<td><!-- TFB1 200 mL -->
 
<ul style="font-size:15px;" align="left">
 
<ul style="font-size:15px;" align="left">
−
<li>  0.59g KOAc </li>
+
<li>  0.59 g KOAc </li>
−
<li> 2.42g RbCl </li>
+
<li> 2.42 g RbCl </li>
−
<li> 0.29g CaCl2.2H<SUB>2</SUB>O </li>
+
<li> 0.29 g CaCl2.2H<SUB>2</SUB>O </li>
−
<li> 1.98g MnCl2.4H<SUB>2</SUB>O </li>
+
<li> 1.98 g MnCl2.4H<SUB>2</SUB>O </li>
 
<li> Adjust to pH 5.8 with 0.2 M acetic acid </li>
 
<li> Adjust to pH 5.8 with 0.2 M acetic acid </li>
−
<li> Add dH<SUB>2</SUB>O to 200mL </li>
+
<li> Add dH<SUB>2</SUB>O to 200 mL </li>
 
<li> Filter sterilize </li>
 
<li> Filter sterilize </li>
 
<li> Store refrigerated at 4°C </li>
 
<li> Store refrigerated at 4°C </li>
 
</ul>
 
</ul>
−
</td><!-- TFB1 200mL END -->
+
</td><!-- TFB1 200 mL END -->
 
 
−
<td><!-- TFB2 200mL -->
+
<td><!-- TFB2 200 mL -->
 
<ul style="font-size:15px;"align="left">
 
<ul style="font-size:15px;"align="left">
−
<li> 0.42g MOPS </li>
+
<li> 0.42 g MOPS </li>
−
<li> 2.21g CaCl2.2H20 </li>
+
<li> 2.21 g CaCl2.2H20 </li>
−
<li> 0.24g RbCl </li>
+
<li> 0.24 g RbCl </li>
 
<li> 30g Glycerol </li>
 
<li> 30g Glycerol </li>
 
<li> Adjust to pH 6.5 with KOH </li>
 
<li> Adjust to pH 6.5 with KOH </li>
−
<li> Add dH<SUB>2</SUB>O to 200mL </li>
+
<li> Add dH<SUB>2</SUB>O to 200 mL </li>
 
<li> Filter sterilize </li>
 
<li> Filter sterilize </li>
 
<li> Store refrigerated at 4°C </li>
 
<li> Store refrigerated at 4°C </li>
 
</ul>
 
</ul>
−
</td><!-- TFB2 200mL END -->
+
</td><!-- TFB2 200 mL END -->
 
           </tr>
 
           </tr>
 
         </tbody>
 
         </tbody>
Line 813: Line 813:
 
<div class="group">
 
<div class="group">
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li>1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C </li>
+
<li>1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37 °C </li>
−
<li>2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.</li>
+
<li>2. Pick a single fresh colony to inoculate 5 mL of YETM medium. Grow over night at 37 °C.</li>
 
<li><b>Do not vortex cells at any time after this point in the procedure</b></li>
 
<li><b>Do not vortex cells at any time after this point in the procedure</b></li>
−
<li>3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C</li>
+
<li>3. Dilute 1 mL of culture into 50 mL YETM medium prewarmed to 37 °C</li>
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li> Grow at 37°C for 2hr with agitation </li>
+
<li> Grow at 37 °C for 2 hours with agitation </li>
 
<li> Volumes can be scaled up 5X and all of the 5mL overnight culture can be used </li>
 
<li> Volumes can be scaled up 5X and all of the 5mL overnight culture can be used </li>
 
</ul>
 
</ul>
 
<li> 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes </li>
 
<li> 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes </li>
−
<li> 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant </li>
+
<li> 5. Centrifuge for 10 minutes at 2,000 rpm at 4 °C. Immediately aspirate off all of the supernatant </li>
−
<li> <b> Do not allow cells to warm above 4°C at any time in this procedure </b> </li>
+
<li> <b> Do not allow cells to warm above 4 °C at any time in this procedure </b> </li>
−
<li> 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette </li>
+
<li> 6. Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette </li>
−
<li> 7. Incubate cells on ice for 5min </li>
+
<li> 7. Incubate cells on ice for 5 minutes </li>
 
<li> 8. Repeat step 5</li>
 
<li> 8. Repeat step 5</li>
−
<li> 9. Resuspend cells in 2mL of ice-cold TFB2 with <b>gentle</b> re-pipetting. Use micropipet tip (plastic)</li>
+
<li> 9. Resuspend cells in 2 mL of ice-cold TFB2 with <b>gentle</b> re-pipetting. Use micropipet tip (plastic)</li>
 
<li> 10. Incubate cells on ice for 15 minutes </li>
 
<li> 10. Incubate cells on ice for 15 minutes </li>
 
<li> Cells may be used for transformation or frozen </li>
 
<li> Cells may be used for transformation or frozen </li>
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li> To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice) </li>
+
<li> To freeze: aliquot cell in 200 μL volumes into prechilled 0.5 mL microfuge tube (on ice) </li>
 
<li> Freeze immediately in liquid nitrogen </li>
 
<li> Freeze immediately in liquid nitrogen </li>
−
<li> Store cells frozen at -80°C </li>
+
<li> Store cells frozen at -80 °C </li>
 
</ul>
 
</ul>
 
</ul>
 
</ul>
Line 846: Line 846:
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
 
<li>1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed. <br>Immedately place on ice for about 5 minutes </li>
 
<li>1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed. <br>Immedately place on ice for about 5 minutes </li>
−
<li>2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.</li>
+
<li>2. Add to 1,5 mL eppendorff on ice: 2-3 μL iGEM plate or 1 μL plasmid or 10 μL ligation.</li>
−
<li>3. Add 100μL of competent cells and mix by gentle re-pipetting</li>
+
<li>3. Add 100 μL of competent cells and mix by gentle re-pipetting</li>
 
<li> 4. Incubate cells on ice for 20-30 minutes </li>
 
<li> 4. Incubate cells on ice for 20-30 minutes </li>
−
<li> 5. Heat shock the cells exactly 90 seconds at 42°C </li>
+
<li> 5. Heat shock the cells exactly 90 seconds at 42 °C </li>
 
<li> 6. Return cells on ice for 2 minutes </li>
 
<li> 6. Return cells on ice for 2 minutes </li>
−
<li> 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking </li>
+
<li> 7. Add 1mL of YETM medium. Incubate at 37 °C for 45-60 minutes with slow gentle shaking </li>
 
<li> 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate  overnight at 37°C</li>
 
<li> 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate  overnight at 37°C</li>
 
  </ul>
 
  </ul>
Line 866: Line 866:
 
<li>1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …) </li>
 
<li>1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …) </li>
 
<li>2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic</li>
 
<li>2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic</li>
−
<li>3. Let the culture grow overnight at 37°C in a shaking incubator</li>
+
<li>3. Let the culture grow overnight at 37 °C in a shaking incubator</li>
−
<li> 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C </li>
+
<li> 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55 °C </li>
−
<li> 5. Keep the tubes at -20°C</li>
+
<li> 5. Keep the tubes at -20 °C</li>
 
</div> <!-- MINIPREP END -->
 
</div> <!-- MINIPREP END -->
 
<br>
 
<br>
Line 964: Line 964:
 
<div class="group">
 
<div class="group">
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li>1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer </li>
+
<li>1. Prepare a 1 % or 2 % electrophoresis agarose gel with 0.5 X TAE buffer </li>
−
<li>2. Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well</li>
+
<li>2. Put 20 µL of sample + 6 µL of marker (1 kb for 1 % gel and 100 pb for 2 %) into the well</li>
 
<li>3. Migration for 30 min at 100 V or 1 hour at 50 V</li>
 
<li>3. Migration for 30 min at 100 V or 1 hour at 50 V</li>
 
<li> 4. Bathe 10 minutes in BET</li>
 
<li> 4. Bathe 10 minutes in BET</li>
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<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
 
<li> Use of DNA Clean up kit for the DNA fragment above 200 pb</li>
 
<li> Use of DNA Clean up kit for the DNA fragment above 200 pb</li>
−
<li> Heat inactivation at 95°C for 10 minutes </li>
+
<li> Heat inactivation at 95 °C for 10 minutes </li>
 
</ul>
 
</ul>
 
  </ul>
 
  </ul>
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  </tr>
 
  </tr>
 
  <tr>
 
  <tr>
−
  <td colspan=2><p>&nbsp;Keep the tubes in ice or at -20°C to prepare the transformation</p></td>
+
  <td colspan=2><p>&nbsp;Keep the tubes in ice or at -20 °C to prepare the transformation</p></td>
 
  </tr>
 
  </tr>
 
         </tbody>
 
         </tbody>
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<div class="group">
 
<div class="group">
 
<ul align="justify" style="font-size:15px;">
 
<ul align="justify" style="font-size:15px;">
−
<li>1. Take 10µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol </li>
+
<li>1. Take 10 µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol </li>
−
<li>2. Plate the solution on selective medium overnight at 37°C</li>
+
<li>2. Plate the solution on selective medium overnight at 37 °C</li>
 
</ul>
 
</ul>
 
</div>
 
</div>

Revision as of 10:42, 9 September 2015

iGEM Toulouse 2015

Experiments & Protocols


Protocols for varroa tests

Protocols for culture tests

Transformation Protocol: RbCl method

Cloning

Protocols for varroa tests

Sampling of varroa

Materials

  • Bee hive
  • Beekeeper suit
  • Gloves
  • Smoker
  • Dry twigs
  • Tweezers
  • Big brush
  • Small brush
  • Petri dishes
    Ø x h = 35 x 15 mm

Methods

  1. Slip beekeeper suit and gloves on and go to bee hive
  2. Fire dry twigs in smoker
  3. Open bee hive and activate smoker to get bees inside the hive
  4. Take a frame out the hive and remove bees with big brush and smoker
  5. Close bee hive
  6. In the lab, put the frame on a table against the wall
  7. With tweezer drill hole into one beehive cell
  8. Remove larvae and look for varroas on larvae and on beehive cell
  9. If there are varroas, take them with a small brush and put them on Petri dishes
  10. Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas
  11. Start again step 7 to 9 until you have enough varroas

Standardization of varroas and sampling

When we take varroas directly from frame, as it is described in protocol “Sampling Varroas”, we have varroas in different phases. In order to have varroas in the same phase it is necessary to add one step and it is important for reproducibility of experiments. With this method we place varroas on adult bees so all varroas will be in phoretic phase.

Materials

  • Bees in box with aeration and glucose
  • Varroas from protocol “Sampling varroas”
  • Gas cylinder of CO2
  • Small brush
  • Tweezers
  • Petri dishes
    Ø x h = 35 x 15 mm

Methods

  1. With small brush take varroas from Petri dish and put them on bees in box through aeration holes
  2. Place the box in a 35°C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator
  3. Take the box out of incubator
  4. Add CO2 from gas cylinder into the box until all bees fall down
  5. Open the box, take a bee with tweezer and look for varroas
  6. When you find a varroa take him with small brush and replace bee in the box
  7. Start again step 5 and 6 until you have enough varroas

Attraction test on varroas

In order to test the attraction effect of butyric acid on varroas an Y test was used, as it is showed with diagram below. Where we place varroas for the test we choose glass pipe because on plastic they could load themselves with electrostatics and die. For butyric acid concentration we choose 4% (V/V) because this is the concentration used in the patent we see, see “Attribution” part.

Materials

  • Pump wich expels air
  • 15 mL Flacon tube
  • Plastic pipe
    Ø = 10 mm
  • Glass T pipe
    Ø = 10 mm, made by a glassworker
  • Plastic separator
  • Carded cotton
  • Absorbent cotton
  • 5mL 4% (V/V) Butyric acid
  • 5mL Water
  • Standardized varroas

Methods

  1. Put a cotton on Petri dish and add 400µL of one acid formic solution
  2. Place three varroas on this Petri dish and close it
  3. Start again step1 and 2 for each formic acid solution and water
  4. Each 30 minutes check if varroas are alive. To do that :
  5. When varroa heads for one side of Glass T tube and covers more than 2 cm test is over and we write down the side choosen by varroa (Butyric acid or Water)
  6. Two tests can be made in the same time thanks to the plastic separator

Mortality test on varroas

To test toxicity of formic acid on varroas we had to choose which concentrations we use. For that we based our thoughts on present treatments. When beekeepers use formic acid for long treatment they place a diffuser at the top of the hive and formic acid concentration was assessed at 200ppm 1 on average that is equivalent to 7.826 mmol.m-3. As gas concentration is difficult to evaluate we calculate the liquid concentration balance thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.

$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$ $$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8.314\times293=19,964 Pa $$

  • PA: partial pressure of A in Pa
  • CA: Concentration of A in air in mol.m-3
  • R: perfect gaz constant = 8.314 J.mol-1.K-1
  • T: temperature in °K

$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$ $$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$

  • CA,eq: equivalent concentration in liquid in mol.L-1
  • HA: Henry's constant = 0,019 Pa.m3mol-1

Materials

  • Petri dishes
    Ø x h = 35 x 15 mm
  • Varroas form “Sampling varroas”
  • Cotton
  • Acid formic solutions :
    • 2 mol.L-1
    • 10 mmol.L-1
    • 1 mmol.L-1
    • 500 µmol.L-1
    • 50 µmol.L-1
  • Water

Methods

  1. Put a cotton on Petri dish and add 400µL of one acid formic solution
  2. Place three varroas on this Petri dish and close it
  3. Start again step1 and 2 for each formic acid solution and water
  4. Each 30 minutes check if varroas are alive. To do that :
    1. Tap on Petri dish and see if varroa move. If yes varroa is alive, if not see below
    2. Look at binocular magnifier if varroas move their feets. If yes varroa is alive, if not varroa is dead
      Remark: A varroa stop moving approximately one or two hours before it dies

Protocols for culture tests

Cytotoxicity tests
    Choice of concentrations

In the begining we tested high and low concentrations and in function of results we adapted concentrations. In the end we worked with these concentrations:

  • Butyric acid : 218 mM, 109 mM, 10.9 mM, 5.45 mM and 1.09 mM
  • Formic acid : 100 mM, 10 mM, 1 mM 500 µM, 100 µM, 50 µM and 25 µM

Materials

  • Optical reader, OPTIMA MARS Analysis
  • 48 wells plates
  • Pre-culture of E. coli BW 25113
  • Acid solutions
  • Medium : LB, M9 15 mM of glucose or 30 mM of glucose

Methods

  1. Add 400 µL of medium in each well
  2. Add 50 µL of pre-culture
  3. Add 50 µL of acid solution
  4. Place the 48 well plate in the optical reader
  5. Adjust parameters on computer.
    Usually we set 250 cycles around 24h so we have an OD measure every six minutes

Remark: Each condition is tested in three replicates

Growth culture

  Culture on erlenmeyers and TubeSpin® Bioreactors

     Inoculation and sampling

Materials

  • Pre-culture of E.Coli BW 25113 in LB
  • Spectrophotometer
  • 1mL Spectrophotometer cuvettes
  • Centrifuge
  • Erlenmeyers
  • TubeSpin® Bioreactors from TPP brand
  • Medium : M9 15 mM of glucose or 30 mM of glucose
  • Incubators at 37°C, 130rpm and without agitation
  • 1.5mL Eppendorf
  • 0.2µm filters

Methods

  1. Add 50 mL of medium on erlenmyer and TubeSpin® Bioreactor
  2. Inoculate from pre-culture to have OD600nm=0.1.
    To do that centrifuge the appropriate volume of pre-culture, then remove LB medium and resuspend sediment with M9 medium to inoculate.
    Remark: This step permits to eliminate substrates from LB medium which could interfere during NMR analysis.
  3. Place erlenmeyers in incubator 37°C 130rpm and TubeSpin® Bioreactor in incubator 37°C without agitation
  4. Sampling every two hours the first day:
    • Take 1mL of culture in 1.5 mL Eppendorf.
      Attention: for TubeSpin® Bioreactor use needle and syringe in order not to let air enter, as possible.
    • Add 100 µL of sample in spectrophotometer cuvette, complete with 900µL water and measure OD600nm with spectrophotometer
    • Centrifuge the rest of samples at 13000 rpm during 3 minutes
    • Filter the supernatant through a 0.2 µm filter and conserve it at -20°C
  5. Days follow sample once a day with method below and plate on Petri dish an appropriate dilution in order to know if bacteria are alive

 NMR analysis

Materials

  • Culture supernatants from -20°C
  • 2.5 mM TSP (Trimethylsilyl propanoic acid) diluted in D2O
  • 0.5 mm NMR tubes
  • 1.5mL Eppendorf
  • Spinners (5mm)
  • 500MHz Bruker Avance Spectrometer

Methods

  1. Add 400 µL of culture supernatant in 1.5mL Eppendorf
  2. Add 100 µL of TSP solution
  3. Place the mix in 0.5 mm NMR tubes
  4. Place NMR tube into spinner, sample is ready to analyse

Culture on 48 wells plates

In order to determine the right concentration of polysaccharide and enzyme of BioSilta kit we have to do several cultures at the same time. So, we use an optical reader and 48 wells plates in order to have lots of test in the same time.

Materials

  • Optical reader, OPTIMA MARS Analysis
  • 48 wells plates
  • Pre-culture of E. coli BW 25113
  • Different concentrations of BioSilta medium
  • For one concentration of BioSilta medium different concentrations of enzyme

Methods

  1. Add 450µL of medium in each well
  2. Add 50µL of pre-culture
  3. Place the 48 well plate in the optical reader
  4. Adjust parameters on computer. We tested culture between one day and ten days

Remark: Each condition is tested almost in two replicates. According to our results we adapt concentrations of Biosilta medium and enzyme, results are exposed in Device part.

Enzyme kinetic

Materials

  • Spectrophotometer
  • BioSilta medium
  • BioSilta enzyme solution named Reagent A (3000U/L)
  • Bradford’s reagent
  • 1.5mL Eppendorf
  • Standard solutions of glucose

Methods

  1. For each standard solutions : in a 1.5 mL Eppendorf sample 10 µL and add 1mL Bradford’s Reagent, wait 20 minutes and measure OD505nm
  2. Plot glucose concentration in function of OD505nm and determine the linear region
  3. Add 1.5 mL of BioSilta medium and 45 µL of reagent A (50 U/L) in an Eppendorf
  4. Sampling every 30 minutes:
    1. Take 10 µL and add 1 mL of Bradford’s reagent in an Eppendorf
    2. Wait 20 minutes
    3. Measure OD505nm
    4. If OD505nm is over linear region dilute sample and measure OD505nm again
  5. Stop sampling when glucose concentration no longer change

Acids production test

In order to test if E.coli produce formic acid and butyric acid with genes we add we made culture test with modified bacteria. We used the same protocol as “Culture on Erlenmeyers and TubeSpin® Bioreactors” with some changes:

  • Volume of culture : 30mL
  • Add Ampicillin at 25µg/mL to have selection pressure
  • The number of samples:
    • Sample at the beginning
    • Sample at the end of the first day
    • Sample after 24h culture and 48h culture

Test of gas concentration

The objective of our device is to produce gas, so we would like to know gas composition of our culture. So, we developed a system in order to recover acids gas.

Materials

  • 4 h culture in 50 mL Falcon in M9 medium with 15 mM of glucose
  • Silicon plugs adapted to 50mL Falcon
  • Needles
  • 0.2 µm filters
  • 10 mL Syringes
  • Neoprene pipes Ø=0.8 mm
  • 10 mM NaHCO3
  • 1.5mL Eppendorf
  • 1mL Sterile cone
  • Incubator 3°C without agitation

Methods

  1. Replace Falcon plug with silicon plug
  2. Adjust fliter on needle and peg it into silicon plug. Do it twice
  3. Adjust neoprene pipe into each filter
  4. Add 700µL of NaHCO3 in an Eppendorf
  5. At the end of first pipe put a sterile cone and immerse it into Eppendorf with NaHCO3
  6. At the end of second pipe put a 10mL syringe
  7. After 24h culture, press 10mL syringe in order to expel gas in NaHCO3 solution
  8. Conserve samples at -20°C before NMR analysis (see protocol foregoing)

Remark 1: We used culture in M9 because with the “Acids production tests” we had data on this medium.
Remark 2: 10 mM NaHCO3 solution was used because pH was 8.3 so it would permit acid gas solubilisation.


Protocols for TPX permeability tests

Preparation of TPX bag

Materials

  • TPX, gas permeable plastic
  • Fusing machine
  • 2 mM Formic acid solution
  • 4 % (V/V) Butyric acid solution

Methods

  1. Prepare plastic bag in sticking on 3 sides over 4 with fusing machine
  2. Add 7mL of appropriate solution in plastic bag
  3. Stick on the last side with fusing machine

Permeability test

To test gas permeability of TPX plastic, we use the same protocol as “Test of gas concentration”. The only change is that no filters were used because the sterility is not necessary.

The device used for the permeability test


Transformation Protocol: RbCl method

Media and solution

YETM 500 mL TFB1 200 mL TFB2 200 mL
  • 2.5 g Yeast Extract
  • 10 g Tryptone
  • 5 g MgSO4.7H2O
  • Adjust pH to 7.5 with KOH
  • For Plates: add 7.5 g of Agar
  • 0.59 g KOAc
  • 2.42 g RbCl
  • 0.29 g CaCl2.2H2O
  • 1.98 g MnCl2.4H2O
  • Adjust to pH 5.8 with 0.2 M acetic acid
  • Add dH2O to 200 mL
  • Filter sterilize
  • Store refrigerated at 4°C
  • 0.42 g MOPS
  • 2.21 g CaCl2.2H20
  • 0.24 g RbCl
  • 30g Glycerol
  • Adjust to pH 6.5 with KOH
  • Add dH2O to 200 mL
  • Filter sterilize
  • Store refrigerated at 4°C

Preparation of Competent Cells

  • 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37 °C
  • 2. Pick a single fresh colony to inoculate 5 mL of YETM medium. Grow over night at 37 °C.
  • Do not vortex cells at any time after this point in the procedure
  • 3. Dilute 1 mL of culture into 50 mL YETM medium prewarmed to 37 °C
    • Grow at 37 °C for 2 hours with agitation
    • Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
  • 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
  • 5. Centrifuge for 10 minutes at 2,000 rpm at 4 °C. Immediately aspirate off all of the supernatant
  • Do not allow cells to warm above 4 °C at any time in this procedure
  • 6. Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
  • 7. Incubate cells on ice for 5 minutes
  • 8. Repeat step 5
  • 9. Resuspend cells in 2 mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
  • 10. Incubate cells on ice for 15 minutes
  • Cells may be used for transformation or frozen
    • To freeze: aliquot cell in 200 μL volumes into prechilled 0.5 mL microfuge tube (on ice)
    • Freeze immediately in liquid nitrogen
    • Store cells frozen at -80 °C

Transformation of Competent Cells

  • 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
    Immedately place on ice for about 5 minutes
  • 2. Add to 1,5 mL eppendorff on ice: 2-3 μL iGEM plate or 1 μL plasmid or 10 μL ligation.
  • 3. Add 100 μL of competent cells and mix by gentle re-pipetting
  • 4. Incubate cells on ice for 20-30 minutes
  • 5. Heat shock the cells exactly 90 seconds at 42 °C
  • 6. Return cells on ice for 2 minutes
  • 7. Add 1mL of YETM medium. Incubate at 37 °C for 45-60 minutes with slow gentle shaking
  • 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C

Minipreps

  • 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
  • 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
  • 3. Let the culture grow overnight at 37 °C in a shaking incubator
  • 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55 °C
  • 5. Keep the tubes at -20 °C

Cloning

First step: Digestion

Both parts have the same antibiotic resistance

Vector Insert

5 µL of miniprep plasmid

10 µL of miniprep plasmid

1 µL of each restriction enzymes

1 µL of each restriction enzymes

2 µL of Green Buffer

2 µL of Green Buffer

10 µL of Milli-Q water

5 µL of Milli-Q water

Incubate 15 minutes at 37°C

The two parts have a different antibiotic resistance

Both parts

5 µL of miniprep plasmid

1 µL of each restriction enzymes

2 µL of Green Buffer

9 µL of Milli-Q water

Incubate 15 minutes at 37°C

Migration and gel extraction

  • 1. Prepare a 1 % or 2 % electrophoresis agarose gel with 0.5 X TAE buffer
  • 2. Put 20 µL of sample + 6 µL of marker (1 kb for 1 % gel and 100 pb for 2 %) into the well
  • 3. Migration for 30 min at 100 V or 1 hour at 50 V
  • 4. Bathe 10 minutes in BET
  • 5. Wash in water for 5 minutes
  • 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit

  • Two ways to inactivate the enzymes for the vector
    • Use of DNA Clean up kit for the DNA fragment above 200 pb
    • Heat inactivation at 95 °C for 10 minutes

Second step: Ligation

Mix Control

10 µL of insert

no insert

4 µL of vector

4 µL of vector

2 µL of 10x T4 buffer

2 µL of 10x T4 buffer

0.5 µL of T4 ligase

0.5 µL of T4 ligase

3.5 µL of Milli-Q water

13.5 µL of Milli-Q water

 Incubate the ligation mix 15 minutes at room temperature (22°C)

 Keep the tubes in ice or at -20 °C to prepare the transformation

Third step: Ligation

  • 1. Take 10 µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
  • 2. Plate the solution on selective medium overnight at 37 °C

References