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Team:INSA-Lyon/Protocols/granules extraction and intein cleavage
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Protocols
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- Competent cells
- Transformation
- DNA extraction
- Digestion
- Ligation
- Measure of temperature and shaking speed influence
- Measure of osmotic pressure influence
- Granules extraction and intein cleavage
- Biofilms quantification
- Extra
Granules extraction and intein cleavage
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(establish thanks to Banki et al.)
- 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 during 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.
