Lisi ultrasonica di 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
- Controllo della temperatura
- for very small to very large samples (µL to litres)
- Trattamento puramente meccanico
- 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 di circa 0,15 in 50 ml di LB (+ 0,2% di glucosio) prima di aggiungere 27 μl di formaldeide (37%) per ml di terreno (concentrazione finale 1%). La reticolazione è stata eseguita con agitazione lenta (100 rpm) a temperatura ambiente per 20 minuti, seguita da spegnimento con 10 ml di glicina 2,5 M (concentrazione finale 0,5 M). Per gli esperimenti di shock termico, E. coli MG1655 è stata coltivata in 65 ml di terreno LB a 30°C fino a una OD600 di circa 0,3. Successivamente, 30 ml di coltura sono stati trasferiti in una fiasca preriscaldata a 43°C e il resto è stato mantenuto a 30°C. La reticolazione e il quenching sono avvenuti come descritto sopra, tranne che per il fatto che le cellule sono state mantenute a 30 o 43°C per 5 minuti prima di un ulteriore agitazione lenta a temperatura ambiente. Le cellule sono state raccolte per centrifugazione e lavate due volte con TBS freddo (pH7,5). Dopo la risospensione in 1 ml di tampone di lisi (10 mM Tris (pH 8.0), 20% saccarosio, 50 mM NaCl, 10 mM EDTA, 10 mg/ml lisozima) e l'incubazione a 37°C per 30 minuti, seguita dall'aggiunta di 4 ml di tampone IP, le cellule sono state sonicate in ghiaccio con 12 pause di 30 sec e 30 sec utilizzando il processore a ultrasuoni Hielscher UP400St al 100% di potenza. Dopo una centrifugazione di 10 minuti a 9000 g, aliquote da 800 μl del surnatante sono state conservate a -20°C. (Waldminghaus 2010)
Overproduction and Purification of Enzymes with an Ultrasonic Probe
Per la sovrapproduzione di proteine marcate con decahistidina (His10), E. coli BL21(DE3) è stata trasformata con i costrutti pET19b. Una precultura di una notte è stata raccolta mediante centrifugazione e l'1% è stato utilizzato per inoculare una coltura di espressione. Le cellule portatrici di pET19mgtB sono state fatte crescere a 22°C fino a raggiungere una densità ottica a 600 nm (OD600) di 0,7. La coltura è stata trasferita a 17°C e indotta con 100 μM di IPTG. Dopo 16 ore, la coltura è stata raccolta mediante centrifugazione a 7.500 × g a 4°C. Le cellule sono state risospese in una soluzione salina tamponata con fosfato (PBS) 50 mM con 0,3 M NaCl a pH 7,4 e disgregate mediante ultrasuoni con un sonotrodo S2 micro-tip all'ultrasuonatore Hielscher UP200St a un ciclo di 0,5 e un'ampiezza del 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.
Gli estratti cellulari grezzi sono stati centrifugati a 15.000 × g e a 4°C per sedimentare i detriti cellulari. Gli estratti chiarificati sono stati caricati su colonne HisTrap FF Crude da 1 ml utilizzando un sistema ÄKTAprime Plus. Gli enzimi sono stati purificati secondo il protocollo del produttore per l'eluizione a gradiente delle proteine His-tagged. Le soluzioni proteiche eluite sono state dializzate due volte con 1.000 volumi di 50 mM PBS, pH 7,4, con 0,3 M NaCl a 4°C. La purificazione è stata analizzata mediante SDS-PAGE al 12%. La concentrazione di proteine è stata determinata con il metodo Bradford utilizzando 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.
Gli ultrasuonatori a sonda, come l'UP400St use the working principle of acoustic cavitation for the efficient lysis of E.coli.
Expression Analysis and Purification of Recombinant Protein using Sonication
Il pellet di E. coli è stato sonicato con l'ultrasuonatore Hielscher UP100H. A questo scopo, il pellet cellulare è stato risospeso in un tampone di lisi raffreddato (50 mM Tris-HCl pH=7,5, 100 mM NaCl, 5 mM DTT, 1 mM PMSF) e raffreddato in ghiaccio per 10 minuti. Quindi, la sospensione cellulare è stata sonicata con 10 brevi raffiche di 10 s seguite da un intervallo di 30 s per il raffreddamento. Infine, i detriti cellulari sono stati rimossi mediante ultracentrifugazione a 4°C per 15 minuti a 14000 rpm. Per confermare l'espressione di rPR, il surnatante è stato fatto scorrere su gel di poliacrilammide al 12% e analizzato mediante SDS-PAGE e Western blotting. La purificazione di rPR è stata effettuata utilizzando la resina Ni2+-NTA (Invitrogen, USA) secondo la guida del produttore. In questa fase è stato utilizzato il metodo di purificazione nativa. La purezza della proteina purificata è stata valutata mediante elettroforesi su gel di poliacrilammide al 12% e successiva colorazione con blu di Coomassie. La concentrazione della proteina purificata è stata misurata con il kit Micro BCA protein assay (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.
Progettazione, produzione e consulenza – Qualità 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 è un'azienda certificata ISO e pone particolare enfasi sugli ultrasuonatori ad alte prestazioni, caratterizzati da tecnologia all'avanguardia e facilità d'uso. Naturalmente, gli ultrasuoni Hielscher sono conformi alla normativa CE e soddisfano i requisiti UL, CSA e RoH.
La tabella seguente fornisce un'indicazione della capacità di lavorazione approssimativa dei nostri ultrasonori:
| Volume di batch | Portata | Dispositivi raccomandati |
|---|---|---|
| piastre multipozzetto / microtiter | n.a. | UIP400MTP |
| CupHorn per fiale o becher | n.a. | Corno a tazza a ultrasuoni |
| reattore a microflusso a ultrasuoni | n.a. | GDmini2 |
| up to 10 vials with 0.5 to 1.5mL | n.a. | VialTweeter |
| 0,5-1,5 mL | n.a. | VialTweeter |
| 1 - 500mL | 10 - 200mL/min | UP100H |
| 10 - 2000mL | 20 - 400mL/min | UP200Ht, UP400St |
| 0,1 - 20L | 0,2 - 4L/min | UIP2000hdT |
| 10 - 100L | 2 - 10L/min | UIP4000 |
| n.a. | 10 - 100L/min | UIP16000 |
| n.a. | più grande | cluster di 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 è stata coltivata in terreno minimo MOPS in un volume totale di 200 ml fino al raggiungimento di un A600 di 0,5. La coltura è stata divisa in colture da 50 ml per il trattamento di stress. Dopo 15 minuti di incubazione con allicina 0,79 mM, diamide 1 mM o dimetilsolfossido (controllo), le cellule sono state raccolte a 4.000g a 4°C per 10 minuti. Le cellule sono state lavate due volte con tampone KPE prima di risospendere il pellet in 700µl di tampone KPE. Per la deproteinizzazione, sono stati aggiunti 300 l di acido solfosalicilico al 10% (w/v) prima della rottura delle cellule mediante ultrasuoni (3 x 1 min; VialTweeter ultrasuoni). I sovranatanti sono stati raccolti dopo centrifugazione (30 minuti, 13.000g, 4°C). Le concentrazioni di acido solfosalicilico sono state ridotte all'1% con l'aggiunta di 3 volumi di tampone KPE. La misurazione del glutatione totale e del GSSG è stata eseguita come descritto sopra. Le concentrazioni di glutatione cellulare sono state calcolate in base a un volume di cellule di E. coli pari a 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)
Particolarità / Cose da sapere
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!
Taglio del DNA a ultrasuoni
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!
Letteratura / Riferimenti
- 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 produce omogeneizzatori a ultrasuoni ad alte prestazioni da laboratorio a dimensioni industriali.