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

From 2013.igem.org

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       <li>The E.coli K12-DH5.1 containing recombinase and Cloramphenicol resistance genes were grown in variable amount of Chloramphenicol LB culture medium.</li>
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       <li>Pre-culture overnight of the strain containing recombinase plasmid at 37°C.</li>
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       <li>After 4h growth at 37°C, the culture was centrifuged for 3 min at 13000 rpm.</li>
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       <li>Culture at 37°C for 4 hours.</li>
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       <li>The cell pellets were resuspended in 500µl <b>Rec buffer</b> containing 20 mM Tris, pH 7.5, 10mM EDT1, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.</li>
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      <li>Centrifuge the culture for 3 min at 13000 rpm.</li>
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       <li>The cells were lysed by sonication for three bursts of 30s.</li>
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       <li>The cell pellets were resuspended in 600µl <b>Rec buffer 1</b><SUP>*</SUP> (for Bxb1 and FimE recombinases) or <b>Rec buffer 2</b><SUP>*</SUP> (for Tp901 and PhiC31 recombinases).</li>
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       <li>After centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinase) was collected and incubated on ice.</li>
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       <li>Sonication of the cells for three bursts of 30s.</li>
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       <li>Centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinases) was collected and incubated on ice.</li>
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   <p class="texte"><b>In vitro recombination test</b><br>
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      <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>
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      <li><b>Rec buffer 2</b> : 20 mM Tris, pH 7.5, 1mM EDTA, 1M NaCl.</li>
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      <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>
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   <p class="texte"><b>In vitro recombination test Bxb1<SUP>*</SUP> recombinase</b><br>
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       <li>Recombination reactions were assembled on ice with recombinase protein extraction solution and a variable amount of solution containing <b>Rec buffer</b> and the gate plasmid.</li>
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       <li>Recombination reactions were assembled on ice with 100 ml<SUP>*</SUP> of  Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 1<SUP>*</SUP> and 60 ng<SUP>*</SUP> the gate plasmid.</li>
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       <li>Reactions were incubated at 37°C for up to 2 hours and heat inactivated at 75°C for 10min.</li>
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       <li>Reactions were incubated at 37°C<SUP>*</SUP>.</li>
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       <li>Reactions were transformed into E.coli K12-DH5.1.</li>
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      <li>Taking sample (20 µl) for different time from 15 min to 5 hours<SUP>*</SUP> and heat inactivated at 75°C for 10min.</li>
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       <li>Spread out the cells on Kanamycine LB agar plate.</li>
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       <li>Reaction taking samples were transformed into E.coli K12-DH5.1.</li>
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       <li>The plate was incubated overnight at 37°C.</li>
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       <li>Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.</li>
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       <li>The plate was incubated overnight at 37°C<SUP>*</SUP>.</li>
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      <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>
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Revision as of 15:23, 4 October 2013

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Notebook

Protocols

In vitro recombinase characterization protocol



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 Bxb1* recombinase

  1. Recombination reactions were assembled on ice with 100 ml* of Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 1* and 60 ng* the gate plasmid.
  2. Reactions were incubated at 37°C*.
  3. Taking sample (20 µl) for different time from 15 min to 5 hours* 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°C*.
  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.


Note: For Xor1 gate characterization, the in vitro recombination test was performed at different Bxb1 concentrations (100µl and 20µl of Bxb1 extraction solution) and different hours of reactions (2 hours and 5 hours).

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