Ultrasonic Lysis of E. Coli
- E. coli bacteria are the most commonly used bacteria in microbiology and biotechnology.
- Ultrasonic cell disruptors deliver reliable and reproducible results for the lysis of E. coli.
- Intense yet precisely controllable cavitation and shear forces result in complete disruption and high extraction yields (e.g. proteins, DNA).
Why is Ultrasonic Cell Disruption of E. coli the Preferred Method?
Ultrasonic homogenizers or probe-type ultrasonicators offer several advantages for E. coli lysis as intense ultrasound efficiently disrupts cell walls and membranes. Probe-type ultrasonicators are widely used for E. coli lysis due to the following reasons:
Protein extraction from E.coli cells is efficiently performed with the ultrasonic probe UP200St
Probe-type ultrasonicator offers many advantages for E. coli lysis. The reliable and precise control over ultrasonic process parameters allow to optimize the operating parameters such as power, duration, and sample handling to achieve the desired results.
Electrophoretic analyses of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.
(study and image: ©Basselet et al. 2008)
Cell Disruption using Ultrasonic Cavitation
Ultrasonic probe-type homogenizers operate with approx. 20,000 cycles per second (at 20kHz) and cause cavitation in liquids or suspensions. Acoustic cavitation microscopic areas of vacuum-like pressures and high temperatures that tear cells apart. Although temperatures may reach several thousand degrees Celsius, cavitation volumes are so small they do not heat the process significantly. Ultrasound generated acoustic cavitation and shear forces perforate or break the cell membrane of bacterial cells such as E.coli. Hielscher ultrasonicators allow the precise control over process parameters such as ultrasonic intensity, amplitude, energy input, and temperature. Thereby, the ultrasonic lysis process can be adjusted optimally to the cell type, cell culture, and process goal.
- precise control of lysis (intensity, amplitude, temperature)
- reliable, reproducible results
- optimal adaption to specific samples
- temperature control
- for very small to very large samples (µL to litres)
- purely mechanical treatment
- user-friendly, safe operation
- linear scale-up from lab to production
VialTweeter for ultrasonic lysis
Ultrasonic Homogenizer vs Other Lysis Techniques
Whilst chemical and enzymatic lysis can be problematic – since chemical lysis can alter protein structures and introduce purification problems and enzymatic lysis requires long incubation times and is not reproducible – ultrasonic disruption is a sophisticated, fast cell disruption method.
Ultrasonic lysis is based on mechanical forces only. No chemicals are added, sonication breaks the cell wall by shear forces. Chemical lysis can alter protein structure and introduce purification problems. Enzymatic disruption requires long incubation times and is not reproducible. Ultrasonic cell disruption of E.coli bacteria cells is fast, simple, reliable and reproducible. That is why Hielscher ultrasonicators are used in biological and biochemical laboratories around the world for sample preparation, pre-ananlytics, in-vitro diagonstics and manifold assays.
General Recommendations for Ultrasonic Lysis
Sonication is the most popular technique for lysing very small, medium and large quantities of cell suspensions – from pico-liters up to 100L/hr (using an ultrasonic flow cell). Cells are lysed by liquid shear and cavitation. DNA is also sheared during sonication, so it is not necessary to add DNase to the cell suspension.
Temperature control during ultrasonic E.coli lysis
By pre-cooling the sample and keeping the sample during sonication on ice, sample thermal degradation of the sample can be easily prevented.
Ideally, samples should be kept ice-cold during lysis, but for most samples it is sufficient if the temperature does not rise above the temperature of culture or tissue source. Therefore it is recommended, to keep the suspension on ice and to sonicate with several short ultrasonics pulses of 5-10 sec and pauses of 10-30 sec. During the pauses, the heat can dissipate in order to re-establish a low temperature. For larger cell samples, various flow cell reactors with cooling jackets are available.
Read here detailed tips and recommendation for a successful ultrasonic lysis!
Protocols for the Ultrasonic Preparation of E. Coli Lysates
Researchers use Hielscher ultrasonic homogenizers for E.coli cell disruption. Below you can find various tested and proven protocols for E.coli lysis using Hielscher ultrasonic homogenizers for various E. coli-related applications.
Cell Growth, Crosslinking and Preparation of E. coli Cell Extracts using Ultrasonics
For SeqA and RNA polymerase ChIP-Chip E. coli MG1655 or MG1655 ΔseqA was grown at 37°C to an OD600 of about 0.15 in 50 ml LB (+ 0.2% glucose) before 27 μl of formaldehyde (37%) per ml medium were added (final concentration 1%). Crosslinking was performed at slow shaking (100 rpm) at room temperature for 20 min followed by quenching with 10 ml of 2.5 M glycine (final concentration 0.5 M). For heat-shock experiments, E. coli MG1655 was grown in 65 ml LB medium at 30°C to an OD600 of about 0.3. Subsequently 30 ml of culture was transferred to a pre warmed flask at 43°C and the remainder kept at 30°C. Crosslinking and quenching was as described above except that cells were kept at 30 or 43°C for 5 min before further slow shaking at room temperature. Cells were collected by centrifugation and washed twice with cold TBS (pH7.5). After resuspension in 1 ml lysis buffer (10 mM Tris (pH 8.0), 20% sucrose, 50 mM NaCl, 10 mM EDTA, 10 mg/ml lysozyme) and incubation at 37°C for 30 min followed by addition of 4 ml IP buffer, cells were sonicated on ice with 12 times 30 sec and 30 sec breaks using the Hielscher ultrasonic processor UP400St at 100% power setting. After centrifugation for 10 min at 9000 g, 800 μl aliquotes of the supernatant were stored at -20°C. (Waldminghaus 2010)
Overproduction and Purification of Enzymes with an Ultrasonic Probe
For overproduction of decahistidine (His10)-tagged proteins, E. coli BL21(DE3) was transformed with pET19b constructs. An overnight preculture was harvested by centrifugation, and 1% was used to inoculate an expression culture. Cells carrying pET19mgtB were grown at 22°C until an optical density at 600 nm (OD600) of 0.7. The culture was transferred to 17°C and induced by 100 μM IPTG. After 16 h, the culture was harvested by centrifugation at 7,500 × g at 4°C. Cells were resuspended in 50 mM phosphate-buffered saline (PBS) with 0.3 M NaCl at pH 7.4 and disrupted by ultrasonication with an S2 micro-tip sonotrode at the Hielscher ultrasonicator UP200St at a cycle of 0.5 and an amplitude of 75%.
The overproduction of decahistidine-tagged GtfC was induced at 37°C at an OD600 of 0.6 with 100 μM IPTG. Cells were then incubated for 4 h, harvested, and lysed as stated above for MgtB.
Crude cell extracts were centrifuged at 15,000 × g and 4°C to sediment the cell debris. The clarified extracts were loaded on 1-ml HisTrap FF Crude columns using an ÄKTAprime Plus system. The enzymes were purified according to the manufacturer’s protocol for gradient elution of His-tagged proteins. Eluted protein solutions were dialyzed twice against 1,000 volumes of 50 mM PBS, pH 7.4, with 0.3 M NaCl at 4°C. The purification was analyzed by 12% SDS-PAGE. The concentration of protein was determined by the Bradford method using Roti-Quant. (Rabausch et al. 2013)
Ultrasonic Extraction of Protein from E. coli Bacteria
A bait protein of interest (in this case, MTV1 of Arabidopsis thaliana) is fused to a GST tag and expressed in BL21 Escherichia coli (E. coli) cells.
- Take one pellet of GST-MTV1 and GST (corresponding to 50 ml bacterial culture) and resuspend each in 2.5 mL ice cold extraction buffer.
- Use an ultrasonicator UP100H (equipped with MS3 microtip-sonotrode for small volumes of approx. 2-5mL) to disrupt the bacterial cells until they are lysed, which is indicated by reduced opacity and increased viscosity. This has to be carried out on ice, and it is recommended to sonicate in intervals (e.g. 10 sec sonicating followed by 10 sec pause on ice and so on). Care has to be taken not to sonicate with too high intensity. If foaming or the formation of a white precipitate is detected, the intensity needs to be lowered.
- Transfer the lysed bacteria solution to 1.5 mL microcentrifuge tubes and centrifuge at 4°C, 16,000 x g for 20 min.
Probe-type ultrasonicators such as the UP400St use the working principle of acoustic cavitation for the efficient lysis of E.coli.
Expression Analysis and Purification of Recombinant Protein using Sonication
The E. coli pellet was sonicated with the Hielscher ultrasonicator UP100H. For this purpose, cell pellet was resuspended in chilled lysis buffer (50 mM Tris-HCl pH=7.5, 100 mM NaCl, 5 mM DTT, 1 mM PMSF) and cooled on ice for 10 min. Then, cell suspension was sonicated with 10 short bursts of 10 s followed by interval of 30 s for cooling. Finally, cell debris was removed by ultracentrifugation at 4°C for 15 min at 14000 rpm. For confirmation of rPR expression, the supernatant was run on 12% polyacrylamide gel and analyzed by SDS-PAGE and Western blotting. Purification of rPR was done using Ni2+-NTA resin (Invitrogen, USA) according to the manufacturer guide. In this stage, native purification method was used. The purity of the purified protein was assessed using electrophoresis on the 12% polyacrylamide gel and subsequent Coomassie blue staining. Purified protein concentration was measured by Micro BCA protein assay kit (PIERCE, USA). (Azarnezhad et al. 2016)
Ultrasonic Homogenizers for E. coli Lysis
Hielscher Ultrasonics designs, manufactures and supplies high-performance ultrasonic homogenizers for the reliable and efficient lysis of E. coli bacteria and other cell types, tissues and cell cultures.
The broad portfolio of ultrasonic probes as well as indirect sonication systems allows us to offer you the ideal ultrasonic tissue homogenizer for your cell disruption and extraction application.
Design, Manufacturing and Consulting – Quality Made in Germany
Hielscher ultrasonicators are well-known for their highest quality and design standards. Smart software, intuitive menu, programmable settings and automatic data protocolling are just a few features of Hielscher ultrasonicators. Robustness and easy operation allow the smooth integration of our ultrasonicators into research and biotech facilities. Even rough conditions and demanding environments are easily handled by Hielscher ultrasonicators.
Hielscher Ultrasonics is an ISO certified company and put special emphasis on high-performance ultrasonicators featuring state-of-the-art technology and user-friendliness. Of course, Hielscher ultrasonicators are CE compliant and meet the requirements of UL, CSA and RoHs.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:
| Batch Volume | Flow Rate | Recommended Devices |
|---|---|---|
| multi-well / microtiter plates | n.a. | UIP400MTP |
| CupHorn for vials or beaker | n.a. | Ultrasonic CupHorn |
| ultrasonic micro-flow reactor | n.a. | GDmini2 |
| up to 10 vials with 0.5 to 1.5mL | n.a. | VialTweeter |
| 0.5 to 1.5mL | n.a. | VialTweeter |
| 1 to 500mL | 10 to 200mL/min | UP100H |
| 10 to 2000mL | 20 to 400mL/min | UP200Ht, UP400St |
| 0.1 to 20L | 0.2 to 4L/min | UIP2000hdT |
| 10 to 100L | 2 to 10L/min | UIP4000 |
| n.a. | 10 to 100L/min | UIP16000 |
| n.a. | larger | cluster of UIP16000 |
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Additional Protocols for Ultrasonic E. coli Lysis
Allicin-modified Proteins in E. coli using an Ultrasonic VialTweeter
Determination of Sulfhydryl Contents by 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) Assay
An E. coli MG1655 overnight culture was used to inoculate MOPS minimal medium (1:100). The culture was grown aerobically until an A600 of 0.4 was reached. The culture was split into three 15-ml cultures for stress treatment. An untreated culture served as a negative control. 0.79 mM allicin (128 μg ml-1) or 1 mM diamide was added to one of the remaining two cultures each. Cultures were incubated for 15 min. 5 ml of each culture were harvested by centrifugation (8,525 × g, 4°C, 10 min). Cells were washed twice with 1 ml of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, stored anaerobically prior to use) and centrifuged (13,000 × g, 4°C, 10 min). Cells were resuspended in lysis buffer (PBS with 6 mM guanidinium HCl, pH 7.4) prior to disruption at 4°C by ultrasonication (VialTweeter ultrasonicator, Hielscher GmbH, Germany) (3 × 1 min). Cell debris was pelleted by centrifugation (13,000 × g, 4 °C, 15 min). The supernatant was transferred to a 3.5-ml QS-macro cuvette (10 mm) with a magnetic stir bar and mixed with 1 ml of lysis buffer. Extinction of the samples was monitored at 412 nm with a Jasco V-650 spectrophotometer equipped with the PSC-718 temperature-controlled cell holder at room temperature. 100μl of a 3 mM dithiobis(2-nitrobenzoic acid) solution were added. Extinction was monitored until it reached saturation. Calculation of thiol concentration was performed using the extinction coefficient ϵ412 = 13,700 M-1 cm-1 for thio-2-nitrobenzoic acid (TNB). Cellular thiol concentrations were calculated based on a volume of E. coli cells of 6.7 × 10-15 liter and a cell density of A600 = 0.5 (equivalent to 1 × 108 cells ml-1 culture). (Müller et al. 2016)
In Vivo Glutathione Determination using an Ultrasonic Cell Crusher
E.coli MG1655 was grown in MOPS minimal medium in a total volume of 200ml until an A600 of 0.5 was reached. The culture was split into 50-ml cultures for stress treatment. After 15 min of incubation with 0.79 mM allicin, 1 mM diamide, or dimethyl sulfoxide (control), cells were harvested at 4,000g at 4°C for 10 min. Cells were washed twice with KPE buffer prior to resuspension of pellets in 700µl of KPE buffer. For deproteination, 300l of 10% (w/v) sulfosalicylic acid were added prior to disruption of cells by ultrasonication (3 x 1 min; VialTweeter ultrasonicator). Supernatants were collected after centrifugation (30 min, 13,000g, 4°C). Sulfosalicylic acid concentrations were decreased to 1% by the addition of 3 volumes of KPE buffer. Measurements of total glutathione and GSSG were performed as described above. Cellular glutathione concentrations were calculated based on a volume of E. coli cells of 6.7×10-15 liter and a cell density of A600 0.5 (equivalent to 1×108 cells ml-1 culture). GSH concentrations were calculated by subtraction of 2[GSSG] from total glutathione. (Müller et al. 2016)
Expression of Human mAspAT in E. coli using an Ultrasonic Homogenizer
The single colony of E. coli BL21 (DE3) harboring the expression vector in 30 mL of Luria-Bertani (LB) medium containing 100μg/mL ampicillin, and then cultivated at 37ºC until the optical density (OD600) reached 0.6. The cells were harvested by centrifugation at 4,000 × g for 10 min, and resuspended in 3L fresh LB medium containing 100μg/mL ampicillin.
Subsequently, protein expression was induced with 1 mM isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) for 20 h at 16ºC. The cells were harvested by centrifugation at 8,000 × g for 15 min and washed with buffer A (20 mM NaH2PO4, 0.5 M NaCl, pH 7.4). Approximated 45g (wet weight) cells were obtained from 3 L culture. After centrifugation, the cell pellets was resuspended in 40 mL (for 1 L culture) ice-cold extraction buffer A, and lysed by ultrasonication at ice-cold temperature using the Hielscher ultrasonic cell crusher UP400St. The cell lysis was centrifuged at 12,000 rpm for 15 min to separate soluble (supernatant) and precipitated (pellet) fractions. (Jiang et al. 2015)
Facts Worth Knowing
E.coli
Escherichia coli (E. coli) is a gram-negative, facultatively anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms). There are a large number of E. coli strains (or subtypes) with diverse characteristics. Most E. coli strains are harmless to humans, e.g. B and K-12 strains which are used commonly for research applications in laboratories. However, some strains are harmful and can cause serious illness.
E. coli plays an important role in modern biological engineering and industrial microbiology since the bacteria is easy to manipulate. Common lab applications which involve often the use of E. coli, e.g. to create recombinant deoxyribonucleic acid (DNA) or to act as a model organism.
E. coli is a very versatile host for the production of heterologous proteins, and manifold protein expression systems are available to produce of recombinant proteins in E. coli. Using plasmids which permit high level expression of protein, genes can be introduced into the bacteria, which enables to produce such proteins in high quantities in industrial fermentation processes.
E.coli are used as cell factories to produce insulin. Further applications include the use of modified E. coli cells to develop and produce vaccines and immobilised enzymes, to produce biofuels, as well as for bioremediation.
The strain K-12 is a mutant form of E. coli that over-expresses the enzyme Alkaline Phosphatase (ALP). This mutation occurs due to a defect in the gene that constantly codes for the enzyme. If a gene produces a product without any inhibition this is known as constitutive activity. This specific mutant form is used for isolation and purification the ALP enzyme.
E. coli bacteria are also widely used as cell factories. Engineered microbes (e.g., bacteria) and plant cells can be used as so-called cell factories. These genetically modified cells produce molecules, chemicals, polymers, proteins, and other substances, which are used for instance in the pharmaceutical, food, and chemical industry. In order to release the molecules produced in the interior of such bioengineered cells, ultrasonic lysis is a common method to disrupt the cell walls and to transfer the target substances into the surrounding liquid. Read more about the lysis of bioengineered cells!
Ultrasonic DNA Shearing
Ultrasonic shear forces are a commonly used method to release molecules, organelles and proteins from the cell interior as well as to break DNA strands into pieces. Acoustic cavitation breaks the cell walls and membranes to extract DNA from cells and generate fragments of about 600 – 800 bp in length, which is ideal for analysis.
Click here to learn more about ultrasonic homogenizers for DNA fragmentation!
Literature / References
- Azarnezhad A., Sharifi Z., Seyedabadi R., Hosseini A., Johari B., Sobhani Fard M. (2016): Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein. Avicenna J Med Biotechnol. 8(4), 2016. 175–181.
- Jiang X., Wang J., Chang H.; Zhou Y. (2016): Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase. BioScience Trends 2016.
- Müller A., Eller J., Albrecht F., Prochnow P., Kuhlmann K., Bandow J.E., Slusarenko A.J., Leichert L.I.O. (2016): Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines. Journal of Biological Chemistry Vol. 291, No. 22, 2016. 11477–11490.
- Rabausch U., Juergensen J., Ilmberger N., Böhnke S., Fischer S., Schubach B., Schulte M., Streit W. R. (2013): Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes. Applied and Environmental Microbiology 79(15), 2013. 4551–4563.
- Sauer M. (2014): MTV1 Pull-down Assay in Arabidopsis. bio-protocol Vol 4, Iss 12, Jun 20, 2014.
- Waldminghaus T., Skarstad K. (2010): ChIP on Chip: surprising results are often artifacts. BMC Genomics 11, 2010. 414.
Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers from lab to industrial size.