Team:INSA Toulouse/contenu/lab practice/notebook/protocols/charac recomb

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   <h2 class="title2">Protocols</h2>
   <h2 class="title2">Protocols</h2>
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   <h3 class="title3">DNA Kit Plate Instructions (iGEM protocol)</h2>
+
   <h3 class="title3"><i>in vitro</i> recombinase characterization protocol</h2>
 +
<br>
 +
  <p class="texte">The different logic gates that have been designed are described in Biological Modules. We imagined a new method for characterizing the logic gates switches with recombinases. This protocol is really easy to apply, can be used to test many recombinases with many sites, avoiding the necessity for lengthy and difficult cloning steps. <br><br>
 +
For easy characterisation of the switching steps without any complicated device, we decided to use the RFP (Red Fluorescent Protein). The gene encoding the RFP protein was directly used as a reporter system, placed under the control of the different promoters of the various logic gates.<br>
 +
Besides the reporter system, each logic gate must be tested with the corresponding recombinase. To save cloning time, we first thought of co-transformation. However, the different tests performed were inconclusive because of the different expression levels of plasmids and incompatibility of pSB1C3 and pSB1A2 derived plasmids. We then decided to test recombination-based switches <i>in vitro</i>.<br><br>
 +
<i>In vitro</i> switch of the designed logic gates avoids the unnecessary DNA assembling steps putting the recombinases on the same plasmid as the logic gate (which is in any case dangerous as the switch can occur during the first cloning step). Recombinases can be produced independently of the logic gates, in a regular <i>E. coli</i> strain and using a plasmid bearing a different antibiotic resistance than the logic gate to be tested. Soluble proteins from the strain carrying the recombinases are extracted and mixed with the plasmid containing the logic gate assembled with RFP. The mix is boiled for a few minutes and further used to transform <i>E. coli</i> (selecting for the antibiotic resistance of the gate, hence the plasmid containing the recombinase is not selected). The bacteria colour gives the result after culture. <br><br>
 +
With this protocol, we have been able to test in due time (two week before the wiki freeze!!) the different recombination based logic gates. The results are available in the <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/recomb">Results part</a><br>.
 +
<b>This protocol can be generalized to any characterization with recombinases with a possible amelioration of the specific recombinase buffer.</b><br>
 +
<br>
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2013/f/fa/Carac_schema_general.jpg" class="imgcontent" />
-
   <p class="texte">Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Transformation Efficiency Kit.
+
<br></br>
-
To use the DNA in the Distribution Kit, follow these instructions: <br><br>
+
 
-
Note: <i>There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µl.</i></p>
+
   <p class="texte"><b>Recombinase overexpression and extraction</b><br>
<div class="list">
<div class="list">
     <ol >
     <ol >
-
       <li>With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.</li>
+
       <li>Pre-culture overnight of the strain containing recombinase plasmid at 37°C.</li>
-
       <li>Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.</li>
+
      <li>Culture at 37°C for 4 hours.</li>
-
       <li>Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.</li>
+
       <li>Centrifuge the culture for 3 min at 13000 rpm.</li>
-
       <li>Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</li>
+
       <li>The cell pellets were resuspended in 600µl <b>Rec buffer 1</b> (for Bxb1 and FimE recombinases) or <b>Rec buffer 2</b> (for Tp901 and PhiC31 recombinases).</li>
-
       <li>Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.</li>
+
       <li>Sonication of the cells for three bursts of 30s.</li>
 +
       <li>Centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinases) was collected and incubated on ice.</li>
     </ol>
     </ol>
<br>
<br>
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<div class="clear"></div>
<div class="clear"></div>
-
  <p class="texte"><i>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.</i><br>
+
<div class="list">
-
Note: <i>There is not enough DNA in each well to perform anything but transformations.</i></p>
+
    <ul >
 +
      <li><b>Rec buffer 1</b> : 20 mM Tris, pH 7.5, 10mM EDTA, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.</li>
 +
      <li><b>Rec buffer 2</b> : 20 mM Tris, pH 7.5, 1mM EDTA, 1M NaCl.</li>
 +
      <li><b>Notes</b> : For overexpression of Tp901 and Bxb1 from Dual Controller plasmid of Bonnet, 20 ng/ml of aTc and 1% of arabinose were added to the culture in order to activate the pTetO and the pBAD promoters.</li>
 +
      </ul>
 +
<br>
 +
<br></div>
 +
<div class="clear"></div>
 +
  <p class="texte"><b><i>in vitro</i> recombination test <b>Bxb1<SUP>1</SUP></b> recombinase</b><br>
 +
 +
<div class="list">
 +
    <ol >
 +
      <li>Recombination reactions were assembled on ice with <b>100 ml<SUP>2</SUP></b> of  Bxb1 recombinase extraction solution and 20 ml of solution containing <b>Rec buffer 1<SUP>3</SUP></b> and <b>60 ng<SUP>4</SUP></b> the gate plasmid.</li>
 +
      <li>Reactions were incubated at <b>37°C<SUP>5</SUP></b>.</li>
 +
      <li>Taking sample (20 µl) for different time <b>from 15 min to 5 hours<SUP>6</SUP></b> and heat inactivated at 75°C for 10min.</li>
 +
      <li>Reaction taking samples were transformed into E.coli K12-DH5.1.</li>
 +
      <li>Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.</li>
 +
      <li>The plate was incubated overnight at <b>37°C<SUP>5</SUP></b>.</li>
 +
      <li>In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.</li>
 +
    </ol>
 +
<br>
 +
<br></div>
 +
<div class="clear"></div>
 +
 +
 +
  <p class="texte">The same protocol is used with different conditions depending on the recombinase(s) used:<br></p>
 +
 +
<table class="tablecontent">
 +
 +
<tr style="background-color:#20a8da; height:50px; color:#ffffff;" >
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><SUP>1 </SUP>Recombinase(s) tested</td>
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>2 </SUP>Buffer</td>
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>3 </SUP>Protein volume (mL)</td>
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>4 </SUP>Plasmid DNA (ng)
 +
</td>
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>5 </SUP>Incubation Temperature (°C)
 +
</td>
 +
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>6 </SUP>Incubation Time
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="border-right:1px solid #e5e6e6;">Bxb1</td>
 +
<td style="border-right:1px solid #e5e6e6;">60</td>
 +
<td style="border-right:1px solid #e5e6e6;">Buffer1</td>
 +
<td style="border-right:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6;">37</td>
 +
<td style="border-right:1px solid #e5e6e6;">15min to 5h</td>
 +
</tr>
 +
<tr style="border-top:1px solid #e5e6e6">
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Tp901</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Buffer2</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">37</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">3h to 10h</td>
 +
</tr>
 +
 +
<tr style="border-top:1px solid #e5e6e6">
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Bxb1 & Tp901
 +
</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100 each</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Buffer2</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">220</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">37</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">3h to 10h</td>
 +
</tr>
 +
 +
<tr style="border-top:1px solid #e5e6e6">
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">PhiC31</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Buffer2</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">30</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">3h to 10h</td>
 +
</tr>
 +
 +
<tr style="border-top:1px solid #e5e6e6">
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">FimE</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Buffer1</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">37</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">3h to 12h</td>
 +
</tr>
 +
 +
<tr style="border-top:1px solid #e5e6e6">
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">PhiC31 & FimE</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">100 each</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Buffer2</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">220</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">30</td>
 +
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">3h to 12h</td>
 +
</tr>
 +
 +
</table>
 +
 +
<br>
 +
<a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/project/references">See protocols references</a>
 +
<br>
</div>
</div>

Latest revision as of 01:35, 5 October 2013

logo


Notebook

Protocols

in vitro recombinase characterization protocol


The different logic gates that have been designed are described in Biological Modules. We imagined a new method for characterizing the logic gates switches with recombinases. This protocol is really easy to apply, can be used to test many recombinases with many sites, avoiding the necessity for lengthy and difficult cloning steps.

For easy characterisation of the switching steps without any complicated device, we decided to use the RFP (Red Fluorescent Protein). The gene encoding the RFP protein was directly used as a reporter system, placed under the control of the different promoters of the various logic gates.
Besides the reporter system, each logic gate must be tested with the corresponding recombinase. To save cloning time, we first thought of co-transformation. However, the different tests performed were inconclusive because of the different expression levels of plasmids and incompatibility of pSB1C3 and pSB1A2 derived plasmids. We then decided to test recombination-based switches in vitro.

In vitro switch of the designed logic gates avoids the unnecessary DNA assembling steps putting the recombinases on the same plasmid as the logic gate (which is in any case dangerous as the switch can occur during the first cloning step). Recombinases can be produced independently of the logic gates, in a regular E. coli strain and using a plasmid bearing a different antibiotic resistance than the logic gate to be tested. Soluble proteins from the strain carrying the recombinases are extracted and mixed with the plasmid containing the logic gate assembled with RFP. The mix is boiled for a few minutes and further used to transform E. coli (selecting for the antibiotic resistance of the gate, hence the plasmid containing the recombinase is not selected). The bacteria colour gives the result after culture.

With this protocol, we have been able to test in due time (two week before the wiki freeze!!) the different recombination based logic gates. The results are available in the Results part
. This protocol can be generalized to any characterization with recombinases with a possible amelioration of the specific recombinase buffer.




Recombinase overexpression and extraction

  1. Pre-culture overnight of the strain containing recombinase plasmid at 37°C.
  2. Culture at 37°C for 4 hours.
  3. Centrifuge the culture for 3 min at 13000 rpm.
  4. The cell pellets were resuspended in 600µl Rec buffer 1 (for Bxb1 and FimE recombinases) or Rec buffer 2 (for Tp901 and PhiC31 recombinases).
  5. Sonication of the cells for three bursts of 30s.
  6. Centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinases) was collected and incubated on ice.


  • Rec buffer 1 : 20 mM Tris, pH 7.5, 10mM EDTA, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.
  • Rec buffer 2 : 20 mM Tris, pH 7.5, 1mM EDTA, 1M NaCl.
  • Notes : For overexpression of Tp901 and Bxb1 from Dual Controller plasmid of Bonnet, 20 ng/ml of aTc and 1% of arabinose were added to the culture in order to activate the pTetO and the pBAD promoters.


in vitro recombination test Bxb11 recombinase

  1. Recombination reactions were assembled on ice with 100 ml2 of Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 13 and 60 ng4 the gate plasmid.
  2. Reactions were incubated at 37°C5.
  3. Taking sample (20 µl) for different time from 15 min to 5 hours6 and heat inactivated at 75°C for 10min.
  4. Reaction taking samples were transformed into E.coli K12-DH5.1.
  5. Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.
  6. The plate was incubated overnight at 37°C5.
  7. In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.


The same protocol is used with different conditions depending on the recombinase(s) used:

1 Recombinase(s) tested 2 Buffer 3 Protein volume (mL) 4 Plasmid DNA (ng) 5 Incubation Temperature (°C) 6 Incubation Time
Bxb1 60 Buffer1 100 37 15min to 5h
Tp901 100 Buffer2 100 37 3h to 10h
Bxb1 & Tp901 100 each Buffer2 220 37 3h to 10h
PhiC31 100 Buffer2 100 30 3h to 10h
FimE 100 Buffer1 100 37 3h to 12h
PhiC31 & FimE 100 each Buffer2 220 30 3h to 12h

See protocols references

Retrieved from "http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb"