Lyse Bacteria by Sonication

<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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You have bacterial cultures and you want to extract something inside of them (e.g., overexpressed protein, plasmid DNA, tRNAs, total lysate, etc.). You’re going to need to lyse this culture. You can use an sonicator to do so, disrupting bacterial walls and membranes using ultrasonic frequencies pumped into the bacterial culture. This protocol shows you how to use a sonicator to mechanically lyse cells.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Sonicator
Sonicator	Q700 Sonicator	QSonica	Q700A-110	$5500	4C to 30C	[link]
Sonicator Probe	Standard Probe, Replaceable Tip, 1/2” (12.7mm) diameter	QSonica	4220	$670	4C to 30C	[link]
Sound Enclosure	Sound Enclosure	QSonica	432B2	$995	4C to 30C	[link]
Lab Jack	Fisherbrand™ Lab Jacks (15cmx15cm)	Fisher Scientific	14-673-51	$484	4C to 30C	[link]

Consumables
15 mL centrifuge tubes	Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case	Corning	430790	$230.44	4C to 30C	[link]
10 mL Luer Lock Syringes	Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL	BD Industrial/Difco	550-80620-PK	$102.40	4C to 30C	[link]
0.45 um syringe filters	PES Syringe Filter, 0.45μm, 30mm, Sterile	CELLTREAT Scientific Products	229749	$84	4C to 30C	[link]

Salts (for Buffers)
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	4C to 30C	[link]
Ammonium Chloride	Ammonium chloride,ACS reagent, ≥99.5%	Sigma-Aldrich	213330-500G	$73.50	4C to 30C	[link]
Sodium Chloride	Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99%	Sigma-Aldrich	746398-1KG	$133.00	4C to 30C	[link]
Magnesium Chloride hexahydrate	BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0%	Sigma-Aldrich	M2670-100G	$50.50	4C to 30C	[link]

Supplements (for Buffers)
Lysozyme	Lysozyme from chicken egg white, protein ≥90%, ≥40,000 units/mg protein, lyophilized powder	Sigma-Aldrich	L6876-1G	$76.90	-25C to -15C	[link]
TCEP-HCl (0.5M; presuspended)	Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL	Thermo Scientific	77720	$175.65	4C to 30C	[link]
cOmplete Protease Inhibitor	cOmplete™ Protease Inhibitor Cocktail, EDTA-Free, Tablets	Roche	11873580001	$472	1C to 4C	[link]

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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[ ] Prepare lysis buffer: Make Protein Purification Buffers and Media.
[ ] Prepare pellets of bacteria expression proteins: Grow and Induce Expression Strains.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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[ ] Prepare Stock Solutions.

Reagent	MW [g/mol]	Amount [g]	Final Volume [mL]	Storage	Needs pH adjustment?	Needs Sterilization?
Stock Solutions
Potassium Hydroxide (1M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (pH=7.6; 1M)	238.3	59.5	250	4C to 30C; dark	yes	no
Ammonium Chloride (1M)	53.49	13.4	250	4C to 30C	no	no
Sodium Chloride (5M)	58.44	73.1	250	4C to 30C	no	no
Magnesium Chloride (1M)	203.3	20.3	100	4C to 30C	no	no
Lysozyme (30 mg/mL)	14320	1	33	-25C to -15C	no	no

[ ] Prepare Lysis Buffer - used to resuspend bacterial pellets for mechanical lysis by sonication. Also chemically lyses cells with lysozyme (G. gallus). Contains protease inhibitor (cOmplete) to slow proteolysis. Make fresh and use the same day!

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	50	1000	5
Ammonium Chloride	100	1000	10
Sodium Chloride	500	5000	10
Magnesium Chloride	10	1000	1
Lysozyme	0.3 mg/mL	30 mg/mL	1
TCEP	1	500	0.2
cOmplete Protease Inhibitor	1 tablet / 50 mL	n/a	2 tablets
water (milliQ)			72.8
Total			100

[ ] Prepare centrifuge and Lysis Buffer.
- [ ] Pre-chill your centrifuge and Lysis Buffer to 4C.
- [ ] Finish Lysis Buffer with TCEP (0.5M) (e.g., add 500 uL TCEP to 1 L Lysis Buffer).
[ ] Resuspend pellets in cold Lysis Buffer.
- [ ] Thaw bacterial pellets on ice.
- [ ] Weigh 1 g dry cell mass per pellet.
- [ ] Resuspend 1 g pellets in 10 mL Lysis Buffer by vortexing.
- Notes - keep lysate cold to prevent proteolysis.
[ ] Lyse cells by sonication.
- [ ] Program your sonicator. We recommend 50% amplitude, 10s on / 10s off.
- Warning - always use a Sound Enclosure during sonication!
- [ ] Place resuspended samples under the sonicator horn in an ice bucket on a lab jack.
- [ ] Adjust your sample height using the sample jack until each sonicator tip is submerged by about one quarter (1/4) of the sample height (e.g., 2.5 mL into a 10 mL sample).
- [ ] Lyse cells by sonication. Deliver 4.5 kJ for every 10 mL sample (e.g., a four-pronged sonicator horn lysing 4x 10 mL samples → 18 kJ).
- Notes - avoid foaming during sonication.
[ ] Clarify lysates by centrifugation and filtration.
- [ ] Centrifuge lysates at 4 krcf / 4C / 45 min.
- [ ] (Meanwhile) for each lysate, assemble a 10 mL Luer lock syringe and 0.22 um syringe filter.
- [ ] Carefully decant each supernatant into a syringe and filter into a fresh 15 mL centrifuge tube. Keep clarified lysate cold (4C).
- Notes - keep going! Don’t take a break!

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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Papers
- Original PURE paper
Cell-free translation reconstituted with purified components
- OnePot PURE
A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

OnePot PURE Cell-Free System | Text Page

<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)

Developers
- Murray Lab