Team:Toulouse/Notebook/Protocols

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   <p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p>  
+
   <div style="margin:0 auto; width:960px;">
 +
  <p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p>
 +
    <ul class="topnav" id="topnav" style="top:15px;">
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   <div id="innercontenthome">
       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
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 +
  <div id="column-left">
 +
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3>
 +
<ul class="menuleft">
 +
  <li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li>
 +
  <li><a href="#select2"><i>E. coli</i> transformation protocol</a></li>
 +
  <li><a href="#select3">Miniprep and alcaline lysis</a></li>
 +
  <li><a href="#select4">Cloning</a></li>
 +
  <li><a href="#select5">Checking of the genetic constructions</a></li>
 +
  <li><a href="#select6"><i>B. subtilis</i> transformation</a></li>
 +
  <li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li>
 +
<li><a href="#select8">Final Tests</a></li>
 +
  </ul>
 +
</div>
 +
<div class="column-right" style="width:75%; float:right;">
-
<div class="Title">Protocols</div>
+
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="http://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p>
-
<br/>
+
-
<br/>
+
-
<div class="Sub_title"> E. coli competent cells <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
-
<br/>
+
-
<TABLE BORDER="1">
+
-
  <TR>
+
-
<TH> Time </TH>
+
-
<TH> Lab equipment </TH>
+
-
<TH> Preparation of media </TH>
+
-
  </TR>
+
-
  <TR>
+
-
<TH> 6 hours </TH>
+
-
<TD>  32mL of 0.1 M CaCl2, glycerol, 4 Falcon tubes, 20 Eppendorf tubes </TD>
+
-
<TD> LB medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl
+
-
<br>
+
-
LB agar medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl + 15g/L agar
+
-
</TD>
+
-
  </TR>
+
-
</TABLE>  
+
-
<div class="Article">
+
<p class="title1" id="select1"><I>E. coli</I> competent cells</p>
 +
<p class="texte"><I> <CENTER> MANIPULATION IN ICE  </CENTER> </I></p>
 +
<p class="texte"><B> Day 0 </B>
 +
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight
<p>
<p>
-
<B> Day 0 </B>
+
<p class="texte"><B> Day 1 </B>
-
</p>
+
<br/>  
-
- Make an Escherichia coli cell culture in LB medium overnight
+
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C
-
<br/>
+
<br/>  
-
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C
+
-
<br/>
+
-
<p>
+
-
<B> Day 1 </B>
+
-
</p>
+
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)
<br/>  
<br/>  
- Centrifuge 10 minutes at 4500 RPM
- Centrifuge 10 minutes at 4500 RPM
<br/>
<br/>
-
<I> <CENTER> MANIPULATION IN ICE FROM THIS STEP </CENTER> </I>
 
- Remove the supernatant
- Remove the supernatant
<br/>
<br/>
-
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2
+
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub>  
-
<br/>
+
-
 Freeze the glycerol
+
-
<br/>
+
-
 Prepare 20 Eppendorfs tubes and put them in ice
+
<br/>
<br/>
- Centrifuge 10 minutes at 4500 RPM
- Centrifuge 10 minutes at 4500 RPM
<br/>
<br/>
-
- Resuspend the pellet in 500µL of 0.1 M CaCl2
+
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub>
<br/>
<br/>
-
- Add glycerol for a final concentration of 15%
+
- Add glycerol to a final concentration of 15%
<br/>
<br/>
- Keep the tubes at -80°C
- Keep the tubes at -80°C
-
<br/>
+
</p>
-
<!-- A compléter et vérifier -->
+
-
</p>
+
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p>
-
<br/>
+
<p class="texte">
-
<div class="Sub_title"> E. coli transformation protocol <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
-
<br/>
+
-
<TABLE BORDER="1">
+
-
  <TR>
+
-
<TH> Time </TH>
+
-
<TH> Lab equipment </TH>
+
-
<TH> Preparation of media </TH>
+
-
  </TR>
+
-
  <TR>
+
-
<TH> 3 hours 30 minutes </TH>
+
-
<TD> LB agar medium plate containing the proper antibiotic (2 plates per transformation), 1 tube of E. coli competent cell per transformation, 1,25mL of LB medium per transformation, water baths at 37°C and 42°C,
+
-
sterile water for kit plate </TD>  
+
-
<TD> LB medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl
+
-
<br>
+
-
LB agar medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl + 15g/L agar
+
-
</TD>
+
-
  </TR>
+
-
</TABLE>  
+
-
<p>
+
- Let the LB agar medium plates dry in a sterile area
- Let the LB agar medium plates dry in a sterile area
<br/>
<br/>
- Thaw out the competent cell aliquotes for about  10 to 20 minutes
- Thaw out the competent cell aliquotes for about  10 to 20 minutes
<br/>
<br/>
-
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA  
+
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA  
<br/>
<br/>
-
<FONT SIZE= 2%> <I> <CENTER> NB: for kit plate, resuspend the well in 10µL of sterile water </CENTER> </I> </FONT> 
+
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i>
-
- Put the tubes 20minutes in the ice
+
- Put the tubes 20 minutes in the ice </i>
<br/>
<br/>
-
- Put the tubes 2 minutes at 42°C in the water bath
+
- Put the tubes 2 minutes at 42°C in the water bath to create the thermic shock
<br/>
<br/>
-
- Put the tubes back in ice immediately to create the thermic shock
+
- Put the tubes back in ice immediately  
<br/>
<br/>
- Add 1mL of LB medium
- Add 1mL of LB medium
<br/>
<br/>
-
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression
+
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression
<br/>
<br/>
-
- Centrifuge for 1 minute at 13 000 RPM
+
- Centrifuge for 1 minute at 13000 RPM
<br/>
<br/>
- Remove the supernatant  
- Remove the supernatant  
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- Resuspend in 250 µL of LB medium
- Resuspend in 250 µL of LB medium
<br/>
<br/>
-
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.
+
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate
-
<br/>
+
  </p>
  </p>
-
<!-- A compléter et vérifier -->
 
   
   
-
<p>
+
<p class="title1" id="select3"> Miniprep and alcaline lysis </p>
-
  <br/>
+
<p class="texte"><B> Day 0 </B>
-
<div class="Sub_title"> Miniprep <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
-
<table width="100%"><tr><td bgColor="#097F09" height="1px"></td> </tr></table>
+
<br/>
<br/>
-
<TABLE BORDER="1">
+
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
-
  <TR>
+
-
<TH> Time </TH>
+
-
<TH> Lab equipment </TH>
+
-
  </TR>
+
-
  <TR>
+
-
<TH> 1 night + 1 hour </TH>
+
-
<TD>  Plates after transformation, culture tubes, LB (5mL x number of tubes), Miniprep kit, cryotubes, Eppendorf tubes </TD>
+
-
</TD>
+
-
  </TR>
+
-
</TABLE>
+
-
<p>
+
-
<B> Day 0 </B>
+
-
</p>
+
-
- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)
+
<br/>
<br/>
-
- Resuspend one colony/culture tube in 5mL LB medium with antibiotic
+
- Let the culture grow overnight at 37°C in a shaking incubator</p>  
-
<br/>
+
 
-
- Leave the culture shakes overnight at 37°C  
+
<p class="texte"><B> Day 1 </B>
-
<br/>
+
<br>- 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.
-
<p>
+
-
<B> Day 1 </B>
+
-
</p>
+
-
- Use a Miniprep kit for each culture tube. Name each final tube with the name, the number of the biobrick and the colony letter. The elution of the DNA (last step) is made with a hot elution buffer or water at 55°C.
+
<br/>
<br/>
- Keep the tubes at -20°C  
- Keep the tubes at -20°C  
-
<br/>
+
<br>
-
- Try the best strain on PCR colony and digestion
+
-
<br/>
+
-
Digestion mix with a 20µL final volume:
+
-
<br/>
+
-
 2µL of plasmid
+
-
<br/>
+
-
 2 µL of green buffer
+
-
<br/>
+
-
 1 µL Enzyme 1
+
-
<br/>
+
-
 1µL Enzyme 2
+
-
<br/>
+
-
 14 µL miliQ water
+
-
<br/>
+
-
- Cryogenise the best colonies in cryotubes : 800 µL of the culture + 200µL of pure glycerol
+
-
<br/>
+
-
- Cryotubes are marked with the bacterium name, the name and the number of the biobrick and the date.
+
-
<br/>
+
-
- Each tube has a number on the top to identify easily in the freezer at -80°C
+
-
<br/>
+
-
</p>
+
-
<!-- A compléter et vérifier -->
+
-
</div>
+
<br/><i>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.
-
<div class="Article">
+
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. </i>
-
    <p>
+
-
  <br/>
+
-
<div class="Sub_title"> Cloning <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
-
<br/>
+
-
<TABLE BORDER="1">
+
-
  <TR>
+
-
<TH> Time </TH>
+
-
<TH> Lab equipment </TH>
+
-
  </TR>
+
-
  <TR>
+
-
<TH> 2 days </TH>
+
-
<TD> Competent cells, BioBricks transformed, Miniprep kit, restriction enzymes, agarose gel (1 or 2%), waterbath, T4 DNA ligase, Eppendorf tubes  </TD>
+
-
</TD>
+
-
  </TR>
+
-
</TABLE>
+
-
<p>
+
-
After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.
+
-
<p>
+
<br>
<br>
-
<B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B>
+
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM
-
<br> 1) Digestion mix
+
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%
 +
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid
 +
</I></p>
 +
 
 +
<p class="title1" id="select4">Cloning </p>
 +
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.
<br>
<br>
-
- 10 µL of miniprep plasmid  
+
<p class="title2">First step</p>
 +
<p class="title3">Both parts have the same antibiotic resistance</p>
 +
<p class="texte"><b>1) Digestion mix</b>
 +
<br> For the vector :
 +
<br>- 5 µL of miniprep plasmid  
<br>
<br>
- 2 µL of each restriction enzymes
- 2 µL of each restriction enzymes
Line 282: Line 265:
- 2 µL of Green Buffer
- 2 µL of Green Buffer
<br>
<br>
-
- 6 µL of water  
+
- 9 µL of Milli-Q water  
<br>
<br>
-
 Incubate 15 minutes at 37°C
+
<br> For the insert :
-
<p>
+
<br>- 10 µL of miniprep plasmid
-
2) Make an electrophoresis
+
<br>
<br>
-
- Prepare 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer
+
- 2 µL of each restriction enzymes
<br>
<br>
-
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well
+
- 2 µL of Green Buffer
<br>
<br>
-
- Migration 30 min at 100V
+
- 4 µL of Milli-Q water
<br>
<br>
-
- The revelation is made in BET (10minutes) and then 5minutes in water
+
- Incubate 15 minutes at 37°C
 +
 
 +
<br><br><b>2) Gel extraction</b>
<br>
<br>
-
<p>
+
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
-
3) Gel extraction with Thermofisher kit
+
<br>
<br>
-
- Remove quickly under UV the DNA fragment from BET to avoid any kind of mutation. Place the fragment in a 1.5mL Eppendorf tube. Make sure to excise as close as possible from the fragment.
+
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well
-
Take a picture under UV before and after the excision.
+
<br>
<br>
-
- Add 200µL of extraction buffer and mix everything
+
- Migration for 30 min at 100 V or 1 hour at 50V
<br>
<br>
-
- Incubate 10-15 minutes at 50-58°C until the gel is dissolved completely.
+
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes
<br>
<br>
-
- Add 200µL of ethanol and mix
+
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit
-
<br>
+
 
-
- Transfer into a column tube and centrifuge 1minute at 14 000 g and remove the liquid
+
<br><br>3) Inactivation of the enzymes for the vector
-
<br>
+
<br>There are two ways to inactivate the enzymes:
-
- Add 200µL of Prewash buffer and centrifuge 1minute at 14 000 g and remove the liquid
+
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
-
<br>
+
<br>- Heat inactivation at 95°C for 10 minutes.</p>
-
- Add 700µL of Wash Buffer and centrifuge 1minute at 14 000 g and remove the liquid. Repeat this step twice.
+
 
-
<br>- Centrifuge the tubes without any liquid 1minute at 14 000 g
+
<p class="title3">The two parts have a different antibiotic resistance</p>
-
- Transfer the column in another 1.5mL Eppendorf tube
+
<p class="texte"><b>1) Digestion mix</b>  
-
<br>- Add 10µL of Elution Buffer and centrifuge 1minute at 14000 g
+
<br>For each part, add:
-
- Remove the column  and keep the tubes at -20°C
+
<br>- 5 µL of miniprep plasmid  
-
<br>
+
-
<P>
+
-
<br>
+
-
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B>
+
-
<br>1') Digestion mix
+
-
<br>- 2 µL of miniprep plasmid  
+
<br>- 1 µL of each restriction enzymes
<br>- 1 µL of each restriction enzymes
<br>- 2 µL of Green Buffer
<br>- 2 µL of Green Buffer
-
<br>- 14 µL of water  
+
<br>- 9 µL of Milli-Q water  
-
<br>Incubate 15 minutes at 37°C  
+
<br>- Incubate 15 minutes at 37°C  
-
<p>
+
 
-
<br>2') To inactivate the enzymes, use a PCR clean up kit or a heat inactivation treatment at 95°C for 10min
+
<p class="texte">
-
<p>
+
<b>2) Inactivation of the enzymes for the vector</b>
-
<br>3') Proceed to the ligation
+
<br>There are two ways to inactivate the enzymes:
-
<p>
+
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
-
<br>
+
<br>- Heat inactivation at 95°C for 10 minutes.</p>
-
<hr>
+
 
-
4) Ligation
+
<p class="title2">Second step</p>
-
<br/>
+
<p class="title3">Ligation</p>
-
- Mix 8µL of insert + 2µL of vector + 2µL of 10x T4 buffer +0.5µL of T4 ligase +H20 qsp 20µL
+
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water
<br/>
<br/>
A control without insert must be made
A control without insert must be made
<br/>
<br/>
-
- Incubate 10 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation
+
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p>
 +
 
 +
<p class="title3">Transformation</p>
 +
<p class="texte">
 +
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.
<br/>
<br/>
-
</p>
+
- Plate the solution on selective medium overnight at 37°C.</p>
-
<p>
+
 
-
5) Transformation
+
<p class="title1 " id="select5">Checking of the genetic constructions </p>
-
<br/>
+
<p class="title2">1) Colony PCR</p>
-
- Take 5µL of the ligation mix for 50µL of competent cells and use the transformation protocol.
+
<p class="texte">
-
<br/>
+
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.
-
- Plate the mix on selective medium overnight
+
-
<br/>
+
-
</p>
+
-
6) Colony PCR
+
-
<br/>
+
-
- Take a colony and add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 5µL of 10µM primer + H20 qsp 50µL.
+
<br/>
<br/>
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.
-
<br/>
+
<br/>The following cycles have been used :
-
</p>
+
<br/>- 94°C - 5 min
-
<p>
+
<br/>- (94°C 45 sec  ; 55°C 45 sec ; 72°C 1min/kb ) *  25 cycles
-
7) Analytic digestion
+
<br/>- 72°C - 5min
-
<br/>
+
<br/>- Then 4°C</p>
-
- Put a colony in 5mL of LB selective medium and wait for 6 hours
+
 
 +
<p class="title2">2) Analytic digestion</p>
 +
<p class="texte">
 +
- Put a colony in 5 mL of LB selective medium and wait for 6 hours
<br/>
<br/>
- Make a purification thanks to the Miniprep kit
- Make a purification thanks to the Miniprep kit
<br/>
<br/>
-
- Mix 5µL of plasmid + 1µL of Fast Digest Green Buffer + 0.3µL of each enzyme +  water qsp 10µL
+
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme +  Milli-Q water qsp 20µL
<br/>
<br/>
-
- Wait 45minutes at 37°C and put the mix on a 1% or 2% gel
+
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p>
-
<br/>
+
 
-
</p>  
+
<p class="title2">3) Sequencing</p>
-
+
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p>
-
<p>
+
 
-
<br/>
+
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p>
-
<div class="Sub_title"> B. subtilis transformation <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
 
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
<p class="texte">
-
<p>
+
<B> Day 0 </B>
<B> Day 0 </B>
-
</p>
+
<br/>- Streak out the <i>B. subtilis</i> strain and plate this on an LB agar plate overnight at 37°C</p>
-
- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C
+
 
-
<p>
+
<p class="texte"><B> Day 1 </B>
-
<B> Day 1 </B>
+
 
-
</p>
+
<br/>- Pick up a nice big colony of <I>B. subtilis </I> strain and drop it in 2ml of completed 1x MC
-
- Pick up a nice big colony and drop it in 2 ml of completed 1x MC
+
<br/>
<br/>
- Grow at 37°C for 5 hours
- Grow at 37°C for 5 hours
<br/>
<br/>
-
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed aeration) and put 6µL of DNA linearized with 1µL of ScaI restriction enzyme
+
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.
<br/>
<br/>
- Grow the cells at 37°C for an additional 2 hours
- Grow the cells at 37°C for an additional 2 hours
<br/>
<br/>
-
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight  
+
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight  
-
<br/>
+
<br/></p>
-
<p>
+
 
-
<b> Preparation of solutions </b>
+
<p class="texte"><b> Preparation of solutions: </b><br>
-
<p> <I> <br> 300 mM Tri-Na Citrate:</I>
+
<I> <br> 300 mM Tri-Na Citrate:</I>
<br>- 0.88 g Tri-Na Citrate
<br>- 0.88 g Tri-Na Citrate
<br>- 10mL MQ water
<br>- 10mL MQ water
-
<p>
+
<br><br><I>Ferric NH4 citrate:</I>
-
<I> <br> Ferric NH4 citrate:</I>
+
<br>- 0.22g Ferric NH4
<br>- 0.22g Ferric NH4
<br>- 10mL MQ water
<br>- 10mL MQ water
-
<p>
+
<br><br><I>10x Competence Medium </I>
-
<I> <br> 10x Competence Medium </I>
+
<br> For 10mL:
<br> For 10mL:
<br>- 1.40g K2HPO4
<br>- 1.40g K2HPO4
Line 412: Line 385:
<br>- 0.2 g Potassium glutamate
<br>- 0.2 g Potassium glutamate
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.
-
 
+
<br><br><I>1x Competence Medium </I>
-
<p>
+
-
<I> <br> 1x Competence Medium </I>
+
<br>- 1.8 mL MQ water
<br>- 1.8 mL MQ water
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)
Line 420: Line 391:
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.
-
<p>
+
 
-
<br/>
+
 
-
<div class="Sub_title"> Tests <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class ="Link">Show more</a></div>
+
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p>
-
<table width="50%"><tr><td bgColor="#097F09" height="1"></td> </tr></table>
+
<p class="texte">
-
<p>
+
<br>- Plate the transformed <i>B. subtilis</i> strain on a selective medium (LB + spectinomycin) overnight
 +
<br>- The obtained clones are then plated on different media:  Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin.
 +
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.
 +
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.
 +
<center><img src="http://2014.igem.org/wiki/images/a/a3/Thr.png" width="400px"></center>
 +
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p>
 +
 
 +
<p class="title1" id="select8">Final Tests</p>
 +
<p class="title2">Chemotaxis test</p>
 +
<p class="texte">
 +
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.
 +
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.
 +
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.
<br>
<br>
-
<B> Plasmid integration test in Bacillus subtilis </B>
+
<center><img src="http://2014.igem.org/wiki/images/b/b1/Installation_1.gif" width="650px"></center>
-
<br>Test of the plasmid integration in Bacillus subtilis genome on the threonine site:
+
<p class="texte">
-
<br>- Plate the transformed Bacillus strain on a selective medium overnight
+
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br>
-
<br>- The obtained clones are then plated on different media: MC(Thr+), MC (Thr-) and LB. These media are equivalent to the ones used for the competence. But in MC(thr-), the casein hydrosylate is not inserted  and replaced by NH4Cl (same amount), threonine is added (5mg/ml). (for the complete protocol, see transformation > B. subtilis)
+
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.</i><br>
-
NB : These media are equivalent to the ones used for the competence except that :
+
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.
-
<br>- For MC(thr-), the casein hydrosylate is not inserted  and replaced by NH4Cl (same amount), threonine is added (5mg/ml). (for the complete protocol, see transformation > B. subtilis)
+
<br>- Let the installation settle for 1 hour at room temperature.
-
<br>-For 500ml of medium :
+
<br>- After an hour, put the volume of the tips on parafilm.
-
<br>-- 450 ml Water
+
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.
-
<br>- 7,5g agar
+
<br>- The plates are then incubated overnight at 37°C.</p>
-
<br>-1,7 ml MgSO4 (1M)
+
-
<br>-Autoclave
+
-
<br>-Add 50ml of 10X MC (+/- thr) and 2.5ml of tryptophane (1%)
+
-
<p>
+
<p class="title2">Binding test</p>
-
<B> Binding test </B>
+
<p class="texte"><i>CBB (Chitin Binding Buffer):</i>
-
<P><I><br>CBB (Chitin Binding Buffer):</I>
+
<br>- 500 mM NaCl
<br>- 500 mM NaCl
<br>- 20 mM Tris-HCl
<br>- 20 mM Tris-HCl
<br>- 1 mM EDTA
<br>- 1 mM EDTA
<br>- 0,05% Triton X-100, 25°C, pH=8
<br>- 0,05% Triton X-100, 25°C, pH=8
 +
</p>
-
<P><I><br>Column activation:</I>
+
<p class="title3">Column activation:</p>
-
<br>- Vortex the beads  
+
<p class="texte">- Vortex the beads  
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube
<br>- Wash with 500 µL of CBB
<br>- Wash with 500 µL of CBB
Line 455: Line 435:
<br>- Remove supernatant
<br>- Remove supernatant
<br>- Repeat the wash
<br>- Repeat the wash
 +
</p>
 +
-
<P><I><br>Bacterial fixation on the chitin beads:</I>
+
<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte">
-
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads  
+
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads  
<br>- Shake during 1h at 4°C
<br>- Shake during 1h at 4°C
-
<br>- Add 500 µL of CBB (washing A)
+
<br>- Add 500µL of CBB (washing A)
<br>- Put the centrifuge tube on a magnetic rack
<br>- Put the centrifuge tube on a magnetic rack
<br>- Wait 30 seconds
<br>- Wait 30 seconds
<br>- Remove supernatant
<br>- Remove supernatant
-
<br>- Add 500 µL of CBB (washing B)
+
<br>- Add 500µL of CBB (washing B)
<br>- Put the centrifuge tube on a magnetic rack
<br>- Put the centrifuge tube on a magnetic rack
<br>- Wait 30 seconds
<br>- Wait 30 seconds
<br>- Remove supernatant
<br>- Remove supernatant
-
<br>- Add 500 µL of CBB to recover the beads directly
+
<br>- Add 500µL of CBB to recover the beads directly
 +
</p>
-
<P><I><br> Bacteria count:</I>
+
<p class="title3">Bacteria count:</p>
-
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates
+
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates
-
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates
+
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates
<br>- Place the plates at 37°C overnight
<br>- Place the plates at 37°C overnight
<br>- Count colonies on different plates
<br>- Count colonies on different plates
 +
</p>
-
<p>
+
<p class="title2">Fungicide test: anti-fungal activities</p>
-
<B> Fungicide test: anti-fungal activities </B>
+
<p class="texte">
-
<p><TABLE BORDER="1">  
+
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br>
-
  <TR>
+
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i>
-
<TH> Time </TH>
+
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
-
<TH> Lab equipment </TH>
+
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.
-
  <TH> Preparation of media </TH>
+
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.
-
  </TR>
+
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i>
-
  <TR>
+
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.
-
<TH> 2 days </TH>
+
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad.
-
<TD> Petri dishes with PDA ¼ medium, fungus, Eppendorf tube, sterile water, parafilm, Thoma cell,  overnight culture of SubtiTree, tips to make the holes, LB + agar </TD> </TD>  
+
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.
-
  <TD> PDA ¼ medium : 2.25 g Agar + 1.95 g PDA (39mg/mL) then complete with 200 mL of water  autoclave </TD> </TD>  
+
</p>
-
</TR>  
+
<p class= "texte">
-
</TABLE>
+
<b> Sap-like Medium (250 mL) (see references)</b>: <br>
-
<p>1- Counting conidia
+
-
<br> <I> /!\ SAFETY : under the biosefty cabinet /!\ </I>
+
2,5g de tryptone
-
<br>- In order to plate an exact number of conidia on PDA plate, first count the conidia  Place a drop of Tween 80 on the fungal plate, let stand (make it roll if possible) and take back the drop in a sterile Eppendorf tube filled with 1ml of water.  
+
-
<br>- Mix and vortex.
+
-
<br>- Place a drop of the mixture in a Thoma cell and count the number of conidia.
+
-
<br><I>NB: You should count at least 5 squares (made of 4x4 squares, marked number 2 below:) and make an average. Multiply this number by 250.000 and you get the number of conodia per ml.</I> 
+
-
<br>- Make dilutions of the solution in order to get the right amount of conidia in each plate: 25.000 /plate and 5000 plate
+
-
<br><I>NB: As we want to spread 200µl per plate you have to get a final concentration of 125.103 conidia / ml (and 25 000 conidia /ml, just make a dilution of the first solution to get this one).</I>
+
-
<br>- Spread on the PDA plate 200 µl of the conidial suspension.
+
-
 
+
-
<p>2- Bacterial plugs
+
-
<br>- Make 2 holes in each PDA plates with a sterile blue tip.  
+
-
<br>- Centrifuge 2ml of an overnight culture of SubtiTree.  
+
-
<br>- Resuspend the pellet in 1ml of LA (less to increase the concentration if you have less tests to perform)
+
-
<br>- Pipet 200µl of the mixture in the hole previously made
+
-
<br> 200µl / fungi / concentration of conidia (4 conditions are tested : Asp 25000, Asp 5000, Glo 25000 and glo 5000)
+
-
<br><I>/!\ Add the LA at the very last minute, just before putting the mixture in the holes to avoid the solidification/!\</I>
+
-
<br>- Place the plates at 30° after putting parafilm around them
+
-
<br>- Do not forget to test the WT and the copper sulphate!
+
-
<p>3- Clean everything
+
-
<br>- Decontaminate the biosafety cabinet (and everything inside) and let the disinfectant into the biosafety cabinet during 30 minutes!!
+
-
 
+
-
 
+
-
<p>
+
-
<B> Chemotaxis test </B>
+
-
<!--à compléter-->
+
 +
<br>1,25g de YE
 +
<br>2,5g de NaCl
 +
<br>Glucose : 1,175g
 +
<br>Fructose : 1,125g
 +
<br>Sucrose : 0,125g
 +
<br>Inositol : 0,084 g
 +
<p class="title2">Fungicide test: <i>in planta</i> assay</p>
 +
<p class="title3">First step</p>
 +
<p class="texte">
 +
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations:  5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.
 +
</p>
 +
<p class="title3">Second step</p>
 +
<p class="texte">
 +
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and  through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.
 +
</p>
 +
<br>
 +
<p class="texte">
 +
<b>References</b><br>
 +
Véronique Amiard, Annette Morvan-Bertrand, Jean-Bernard Cliquet, Jean-Pierre Billard,
 +
Claude Huault, Jonas P. Sandström, and Marie-Pascale Prud’homme. <b>Carbohydrate and amino
 +
acid composition in phloem sap of Lolium perenne L. before and after defoliation</b>. Can. J. Bot.
 +
Vol. 82: 1594–1601, 2004.</p>
 +
</div>
 +
<div class="clear"></div>
       </div>
       </div>
-
    </div>
 
   </div>
   </div>
</body>
</body>

Latest revision as of 03:37, 18 October 2014