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

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<h5 style="text-indent:20px">Measure of fluorescence</h5>
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<h5 style="text-indent:20px">Granules extraction and intein cleavage</h5>
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<ul style="margin-left:-65px;">
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<li>Introduce 5 mL of half LB in a sterile tube</li>
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<li>Resuspend bacteria in 0,5 mL of sterile water. Vortex</li>
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<i>(establish thanks to Banki 2005)</i>
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<li>Add 10 µL of the mixture in the LB tube. Vortex</li>
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<li>Incubate 24 h in a waterbath at the temperature and the shaking speed wished</li>
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<li>Lysis buffer (pH=8,5)
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<li>Measure of the fluorescence : input wavelenght 490 nm, output one 510 nm.</li>
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<li>- 20mM Tris</li>
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<li>Dilute 250 µL of culture in 750 µL of water. Measure the biomass (absorbance 600 nm)</li>
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<li>- 20nM Bis</li>
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<li>- 50nM NaCl</li>
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<li>- 1mM DTT</li>
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<li>- 2mM EDTA</li>
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<li>- 0,25 mg/100 mL lysozyme</li>
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Wash buffer (pH=8,5)
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- 20mM Tris
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- 20nM Bis
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- 50nM NaCl
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- 1mM DTT
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- 2mM EDTA
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Cleavage buffer (pH=6)
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- 20mM Tris
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- 20nM Bis
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- 50nM NaCl
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- 1mM DTT
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- 2mM EDTA
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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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- Induction of the fusion proteins (according to the regulation system chosen)
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- Incubation during 4 additional hours
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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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- Sonication of the cells to disrupted them, at 4°C
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- Centrifugation at 14000 g for 10-30 min at 4°C.
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- Discard the surpernatant  and resuspend the cells in a wash buffer
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- Centrifugation at 14000 g for 10-30 min at 4°C.
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This washing step will be repeated as necessary
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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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- Resuspende the cells in the cleavage buffer at temperature room during to allow the cleavage
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- The evolution of the cleaving process can be follow taking sample
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A completer
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Revision as of 08:14, 26 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


      (establish thanks to Banki 2005)
    • 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 - Resuspende the cells in the cleavage buffer at temperature room during to allow the cleavage - The evolution of the cleaving process can be follow taking sample A completer

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