Team:INSA-Lyon/Protocols/granules extraction and intein cleavage

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<h5 style="text-indent:20px">Granules extraction and intein cleavage</h5>
<h5 style="text-indent:20px">Granules extraction and intein cleavage</h5>
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<ul style="margin-left:50px;">
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  <i>(establish thanks to <a href="https://2010.igem.org/Team:INSA-Lyon/Project/References#et1">Banki et al.</a>)</i>
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</ul>
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  <i>(establish thanks to Banki 2005)</i>
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<ul style="margin-left:-65px;">
<ul style="margin-left:-65px;">
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<li>Lysis buffer (pH=8,5)
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<li><u>Lysis buffer (pH=8,5)</u>
<li>- 20mM Tris</li>
<li>- 20mM Tris</li>
<li>- 20nM Bis</li>
<li>- 20nM Bis</li>
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<li>- 0,25 mg/100 mL lysozyme</li>
<li>- 0,25 mg/100 mL lysozyme</li>
</li>
</li>
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<br>
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<li>Wash buffer (pH=8,5)
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<li><u>Wash buffer (pH=8,5)</u>
<li>- 20mM Tris</li>
<li>- 20mM Tris</li>
<li>- 20nM Bis</li>
<li>- 20nM Bis</li>
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<li>- 2mM EDTA</li>
<li>- 2mM EDTA</li>
</li>
</li>
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<br>
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<li>Cleavage buffer (pH=6)
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<li><u>Cleavage buffer (pH=6)</u>
<li>- 20mM Tris</li>
<li>- 20mM Tris</li>
<li>- 20nM Bis</li>
<li>- 20nM Bis</li>
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<li>- 2mM EDTA</li>
<li>- 2mM EDTA</li>
</li>
</li>
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<br>
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- Growth of the cells during 30h in LB medium supplemented with 2% sodium lactate used as a carbone source for PHB synthesis
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<li>- Growth of the cells during 30h in LB medium supplemented with 2% sodium lactate used as a carbone source for PHB synthesis</li>
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- Induction of the fusion proteins (according to the regulation system chosen)
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<li>- Induction of the fusion proteins (according to the regulation system chosen)</li>
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- Incubation during 4 additional hours
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<li>- Incubation during 4 additional hours</li>
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- 1mL of sample is suspended in 300mL lysis buffer at pH 8,5. At this pH, the protein intein is stable and it is necessary to wash the granule and as consequency the interest proteins.
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<li>- 1mL of sample is suspended in 300mL lysis buffer at pH 8,5. At this pH, the protein intein is stable and it is necessary to wash the granule and as consequency the interest proteins.</li>
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- Sonication of the cells to disrupted them, at 4°C
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<li>- Sonication of the cells to disrupted them, at 4°C</li>
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- Centrifugation at 14000 g for 10-30 min at 4°C.
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<li>- Centrifugation at 14000 g for 10-30 min at 4°C.</li>
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- Discard the surpernatant  and resuspend the cells in a wash buffer
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<li>- Discard the surpernatant  and resuspend the cells in a wash buffer</li>
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- Centrifugation at 14000 g for 10-30 min at 4°C.
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<li>- Centrifugation at 14000 g for 10-30 min at 4°C.</li>
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This washing step will be repeated as necessary
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<li><i>This washing step will be repeated as necessary</i></li>
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<br><br>
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- The final wash with a cleavage buffer, at pH=6 which allow to initiate the intein self–cleavage reaction
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- Centrifugation at 14000 g for 10-30 min at 4°C to ensure the homogenous pH throughout the pellet
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<li>- The final wash with a cleavage buffer, at pH=6 which allow to initiate the intein self–cleavage reaction</li>
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- Resuspende the cells in the cleavage buffer at temperature room during to allow the cleavage
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<li>- Centrifugation at 14000 g for 10-30 min at 4°C to ensure the homogenous pH throughout the pellet</li>
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- The evolution of the cleaving process can be follow taking sample  
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<li>- Resuspend the cells in the cleavage buffer at temperature room to allow the cleavage</li>
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A completer
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<li>- The evolution of the cleaving process can be followed by taking sample and analyse of the protein concentration of the supernatant.</li>
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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Latest revision as of 09:56, 27 October 2010




Protocols


Choose a protocol to read its description :

  1. Competent cells
  2. Transformation
  3. DNA extraction
  4. Digestion
  5. Ligation
  6. Measure of temperature and shaking speed influence
  7. Measure of osmotic pressure influence
  8. Granules extraction and intein cleavage
  9. Biofilms quantification
  10. Extra






    Granules extraction and intein cleavage


    • Lysis buffer (pH=8,5)
    • - 20mM Tris
    • - 20nM Bis
    • - 50nM NaCl
    • - 1mM DTT
    • - 2mM EDTA
    • - 0,25 mg/100 mL lysozyme

    • Wash buffer (pH=8,5)
    • - 20mM Tris
    • - 20nM Bis
    • - 50nM NaCl
    • - 1mM DTT
    • - 2mM EDTA

    • Cleavage buffer (pH=6)
    • - 20mM Tris
    • - 20nM Bis
    • - 50nM NaCl
    • - 1mM DTT
    • - 2mM EDTA

    • - Growth of the cells during 30h in LB medium supplemented with 2% sodium lactate used as a carbone source for PHB synthesis
    • - Induction of the fusion proteins (according to the regulation system chosen)
    • - Incubation during 4 additional hours
    • - 1mL of sample is suspended in 300mL lysis buffer at pH 8,5. At this pH, the protein intein is stable and it is necessary to wash the granule and as consequency the interest proteins.
    • - Sonication of the cells to disrupted them, at 4°C
    • - Centrifugation at 14000 g for 10-30 min at 4°C.
    • - Discard the surpernatant and resuspend the cells in a wash buffer
    • - Centrifugation at 14000 g for 10-30 min at 4°C.
    • This washing step will be repeated as necessary


    • - The final wash with a cleavage buffer, at pH=6 which allow to initiate the intein self–cleavage reaction
    • - Centrifugation at 14000 g for 10-30 min at 4°C to ensure the homogenous pH throughout the pellet
    • - Resuspend the cells in the cleavage buffer at temperature room to allow the cleavage
    • - The evolution of the cleaving process can be followed by taking sample and analyse of the protein concentration of the supernatant.


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