A Procedure for Bacterial Harvesting and Mechanical Lysis

Samenvatting

Source: Mercier, N., et al. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)

This video demonstrates a step-by-step procedure for harvesting bacterial cells and mechanically lysing them to release intracellular contents.

Protocol

1. Bacteria harvesting

CAUTION: This step involves handling of pathogenic bacteria.

1. Grow one colony of strains carrying either pCN51-P3-MS2-sRNA or pCN51-P3-MS2 plasmids in 3 mL of Brain Heart Infusion (BHI) medium supplemented with erythromycin (10 µg/µL) in duplicates.
2. Dilute each overnight culture in 50 mL (≈1/100) of fresh BHI medium supplemented with erythromycin (10 µg/µL) to reach an OD_600nm of 0.05. Use 250 mL sterilized flasks (5:1 flask-to-medium ratio).
   NOTE: Medium and growth conditions should be set according to the expression pattern of the studied sRNA.
3. Grow cultures at 37°C with shaking at 180 rpm for 6 h.
4. Transfer each culture into a 50 mL centrifuge tube.
5. Centrifuge at 2,900 x g during 15 min at 4°C. Discard the supernatant.
6. Keep pellets on ice and directly perform mechanical cell lysis or freeze and store pellets at -80 °C.

2. Mechanical cell lysis

CAUTION: Following steps must be performed on ice and buffers must be at 4 °C. Use gloves and take all precautions to protect samples from RNases.

1. Resuspend pellets (step 1.6) in 5 mL of Buffer A (150 mM KCl, 20 mM Tris-HCl pH 8, 1 mM MgCl₂ and 1 mM DTT).
2. Transfer the resuspended cells in 15 mL centrifuge tubes with 3.5 g of silica beads (0.1 mm).
3. Insert tubes in a mechanical cell lysis instrument. Run a cycle of 40 s at 4.0 m/s.
   NOTE: If one cycle is not enough to break cells, let the device cool for 5 min while keeping samples on ice. Then, repeat another cycle of 40 s at 4.0 m/s. The efficiency of cell lysis can be tested by plating the supernatant on BHI-agar plate.
4. Centrifuge at 15,700 x g for 15 min. Recover the supernatant and keep it on ice.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
15 mL centrifuge tubes	Falcon	352070
50 mL centrifuge tubes	Falcon	352051
250 mL culture flask	Dominique Dutscher	2515074	Bacterial cultures
BHI (Brain Heart Infusion) Broth	Sigma-Aldrich	53286	Growth medium
Erythromycin (ery)	Sigma-Aldrich	Fluka 45673	Growth medium
Lysing Matrix B Bulk	MP Biomedicals	6540-428	Mechanical lysis
RNAPro Solution	MP Biomedicals	6055050	Mechanical lysis
FastPrep device	MP Biomedicals	116004500	Mechanical lysis
Spectrophotometer Genesys 20	ThermoFisher Scientific	11972278	Bacterial cultures