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Preparation of Bacillus subtilis Cell Lysates and Membranes

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In this protocol

Original research article

A brief version of this protocol appeared in:
Molecular Microbiology
Jun 2013

Abstract

A common feature of every eukaryotic and prokaryotic cell is that they exhibit a plasma membrane. In Bacillus subtilis (B. subtilis) roughly 25% of all proteins are putative trans- or membrane associated proteins. Here we describe a relatively simple method to separate and prepare membrane and cytosolic proteins by ultra-centrifugation.

Materials and Reagents

  1. Bacillus subtilis (B. subtilis)
  2. Glycerol (Carl Roth, catalog number: 7530.4 )
  3. Tris (J.T.Baker®, catalog number: 1414 )
  4. NaCl (AppliChem GmbH, catalog number: A3597.5000 )
  5. MgCl2 (Merck KGaA, catalog number: 1.05833.1000 )
  6. Proteinase inhibitor (Roche Diagnostics, catalog number: 0 4693159001 )
  7. DNAse I (Roche Diagnostics, catalog number: 10104159001 )
  8. Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
  9. Casein hydrolysate (Oxoid Limited, catalog number: LP0041 )
  10. Buffer A (see Recipes)
  11. Casein Hydrolysate (CH-medium) (see Recipes)
  12. Solution G (see Recipes)

Equipment

  1. Glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277 )
  2. French press homogenizer (Glen Mills, French press G-MTM )
  3. Ultra-centrifuge (Beckman Coulter, model: optimaTM XPN-100 )
  4. Ti-70 Rotor (Beckman Coulter)
  5. FastPrep tissue homogenizer (MP Biomedicals, model: 116004500 )
  6. Refrigerated centrifuge (e.g. Beckman Coulter, model: Avanti-J25 )
  7. Acrodisc® syringe filters (a pore size of 0.2 µm) (Pall, catalog number: 4652 )

Procedure

  1. Inoculate B. subtilis to an OD600 of 0.1 in appropriate medium [e.g. CH, lysogeny broth (LB), Spizizen minimal medium (SMM); here we used 50 ml cultures in CH-medium].
  2. Grow cells to respective OD600 (here we used an OD600 of 4, cells growing at 37 °C; 150 rpm in 250 ml flasks with baffles).
  3. Harvest cells by centrifugation (10,000 x g; 15 min; 4 °C).
  4. Discard supernatant and resuspend cells in 1/5 volumes of original culture at 4 °C in pre-cooled Buffer A.
  5. Centrifuge again (10,000 x g; 15 min; 4 °C).
  6. Discard supernatant and resuspend cells in 5-8x times volume of the cell pellet in Buffer A at 4 °C supplemented with proteinase inhibitor and DNase I. Use concentrations as specified by the manufacturer.  
  7.  Disrupt cells
    Note: If cell disruption is critical, the cell suspension can be incubated with lysozyme (50 μg/ml) on ice for 30-120 min prior to cell disruption, check microscopically.
    1. Small cell volumes (1 ml in reaction tubes) can be disrupted in a FastPrep tissue homogenizer for 30 sec at 6.5 m/s with diameter 0.2-0.3 mm glass beads and cooling for 5 min on ice between runs (minimum 5 runs).
    2. Larger cell volumes can be disrupted in a French press homogenizer at 125 MPa and 3-5 passes. Cool sample on ice for 5 min between every pass. Note that only use of a French Press system will yield inside-out vesicles.
  8. Remove cell debris by centrifugation (12,000 x g; 15 min; 4 °C).
  9. Collect the supernatant (cell lysate) and centrifuge it at ≥ 200,000 x g; 4 °C and 60-90 min (e.g. in a Beckman-Coulter® Ti-70 rotor at 45,000 rpm).
    Note: Longer centrifugation time results in a better separation of membranes at high protein concentrations.
  10. The supernatant represents the cytoplasmatic fraction; the pellet contains B. subtilis membranes (lipids, trans-membrane proteins and membrane associated proteins).
    Note: Resuspend the pellet in an appropriate buffer, e.g. Buffer A.

Recipes

  1. Buffer A
    50 mM Tris.HCl (pH 7.5)
    150 mM NaCl
    5 mM MgCl2
    10% Glycerol (v/v)
  2. CH-medium
    50 ml solution G (see below)
    0.05 ml 0.1 M CaCl2
    0.02 ml 1 M MgSO4.7H2O
    0.1 ml 0.0475 M MnSO4
    0.5 ml 2 mg x ml-1 tryptophan
    Sterilize by filtering using Acrodisc® syringe filters; prepare freshly
  3. Solution G
    25.0 g casein hydrolysate
    9.2 g L-glutamic acid
    3.125 g L-alanine
    3.48 g L-asparagine
    3.4 g KH2PO4
    1.34 g NH4Cl
    0.27 g Na2SO4
    0.24 g NH4NO3
    2.45 mg FeCl3.6H2O
    ddH2O 2.35 L and adjust pH to 7.0 by using 10 N NaOH
    Sterilize by autoclaving; stored at 4 °C

Acknowledgments

This protocol is adapted from Bach and Bramkamp (2013).

References

  1. Bach, J. N. and Bramkamp, M. (2013). Flotillins functionally organize the bacterial membrane. Mol Microbiol 88(6): 1205-1217.
How to cite: Bach, J. N. and Bramkamp, M. (2014). Preparation of Bacillus subtilis Cell Lysates and Membranes. Bio-protocol 4(2): e1025. DOI: 10.21769/BioProtoc.1025.
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