Cell Lysis of BL21 Cells by Ultrasonication
BL21 Cells for Protein Expression
BL21 cell is a chemically competent E. coli bacterial strain suitable for transformation and high level protein expression using a T7 RNA polymerase-IPTG induction system. BL21 cells allow high-efficiency protein expression of any gene that is under the control of a T7 promoter. The E. coli strain BL21(DE3) is a T7 RNA polymerase-based protein production strain combined with T7 promoter-based expression vectors and is widely applied in laboratories and industry to produce recombinant proteins. In BL21(DE3), expression of the gene encoding the recombinant protein is transcribed by the chromosomally encoded T7 RNA polymerase (T7 RNAP), which transcribes eight times faster than conventional E. coli RNAP. This makes the strain BL21(DE3) highly efficient and turns its into one of the most preferred protein expression cell systems.
Protocol for Ultrasonic Lysis and Protein Extraction from BL21 Cells
Cell lysis of BL21 cells is mostly performed using ultrasonication in combination with sodium lauroyl sarcosinate (also known as sarkosyl) as lysis buffer. The advantages of ultrasonic cell disruption and protein extraction lie in the reliability, reproducibility as well as simple, safe and rapid operation of ultrasonicators. The protocol below gives a step-by-step direction for ultrasonic BL21 cell lysis:
- In order to remove the chaperone proteins, BL21 bacterial pellets were resuspended in 50 ml of ice cold Sodium Tris-EDTA (STE) buffer (consisting in 10 mM Tris-HCL, pH 8.0, 1 mM EDTA, 150 mM NaCl supplemented with 100 mM PMSF).
- The, 500 ul of lysozyme (10 mg/ ml) are added and the cells are incubated on ice for 15 min.
- Afterwards, 500 ul of DTT and 7 ml of sarkosyl (10% (w/v) made up in STE buffer) are added.
- It is essential to keep all purification buffers ice-cold and to maintain the samples on ice all time. All purification steps should be carried out in the cold room if possible.
- For ultrasonic lysis and protein extraction, the samples are sonicated in the VialTweeter MultiSample Ultrasonicator for 4 x 30 sec at 100% amplitude with a 2 min interval between each sonication. Alternatively, a probe-type ultrasonic homogenizer with micro-tip e.g., UP200Ht with S26d2 (3 x 30 sec, 2 min. pause between ultrasonic cycles, 80% amplitude) can be used.
- For further purification steps, samples must be kept on ice or alternatively stored at -80°C until further processing.
Ultrasonic Lysis under Prescise Temperature Control
The precise and reliable temperature control is crucial when handling biological samples. High temperatures initiate thermally-induced protein degradation in samples.
As all mechanical sample preparation techniques, sonication creates heat. However, the temperature of the samples can be well controlled when using the VialTweeter. We present you various options to monitor and control the temperature of your samples whilst preparing them with the VialTweeter and VialPress for analysis.
- Monitoring the sample temperature: The ultrasonic processor UP200St, which drives the VialTweeter, is equipped with an intelligent software and a pluggable temperature sensor. Plug the temperature sensor into the UP200St and insert the tip of the temperature sensor in one of the sample tubes. Via digital coloured touch-display, you can set in the menu of the UP200St a specific temperature range for your sample sonication. The ultrasonicator will automatically stop when the max temperature is reached and pause until the sample temperature is down to the lower value of the set temperature ∆. Then the sonication starts automatically again. This smart feature prevents heat-induced degradation.
- The VialTweeter block can be pre-cooled. Put the VialTweeter block (only the sonotrode without transducer!) into the fridge or freezer to pre-cool the titanium block helps to postpone temperature rise in the sample. If possible, the sample itself can be pre-cooled too.
- Use dry ice to cool during sonication. Use a shallow tray filled with dry ice and place the VialTweeter on the ice so that heat can rapidly dissipate.
Customers worldwide use the VialTweeter and VialPress for their daily sample preparation work in biological, biochemical, medical and clinical laboratories. The intelligent software and temperature control of the UP200St processor, temperature is reliably controlled and heat-induced sample degradation avoided. Ultrasonic sample preparation with the VialTweeter and VialPress delivers highly reliable and reproducible results!
Find the Optimal Ultrasonic Disruptor for your Lysis Application
Hielscher Ultrasonics is long-time experienced manufacturer of high-performance ultrasonic cell disrupters and homogenizers for laboratories, bench-top and industrial scale systems. Your bacterial cell culture size, your research or production goal and the volume of cell to process per hour or day are essential factors to find the right ultrasonic cell disruptor for your application.
Hielscher Ultrasonics offers various solutions for the simultaneous sonication of multi-samples (up to 10 vials with the VialTweeter) and mass samples (i.e., microtiter plates / ELISA plates with the UIP400MTP), as well as the classic probe-type lab ultrasonicator with different power levels from 50 to 400 watts to fully industrial ultrasonic processors with up to 16,000watts per unit for commercial cell disruption and protein extraction in large production. All Hielscher ultrasonicators are built for the 24/7/365 operation under full load. Robustness and reliability are core features of our ultrasonic devices.
All digital ultrasonic homogenizers are equipped with smart software, coloured touch display and automatic data protocolling, which make the ultrasonic device into a convenient work tool in lab and production facilities.
Let us know, what kind of cells, what volume, with what frequency and with what target you have to process your biological samples. We will recommend you the most suitable ultrasonic cell disruptor for your process requirements.
The table below gives you an indication of the approximate processing capacity of our ultrasonic systems from compact hand-held homogenizers and MultiSample Ultrasonicators to industrial ultrasonic processors for commercial applications:
|Batch Volume||Flow Rate||Recommended Devices|
|96-well / microtiter plates||n.a.||UIP400MTP|
|10 vials à 0.5 to 1.5mL||n.a.||VialTweeter at UP200St|
|0.01 to 250mL||5 to 100mL/min||UP50H|
|0.01 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||UIP4000hdT|
|n.a.||10 to 100L/min||UIP16000|
|n.a.||larger||cluster of UIP16000|
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Literature / References
- Cheraghi S.; Akbarzade A.; Farhangi A.; Chiani M.; Saffari Z.; Ghassemi S.; Rastegari H.; Mehrabi M.R. (2010): Improved Production of L-lysine by Over-expression of Meso-diaminopimelate Decarboxylase Enzyme of Corynebacterium glutamicum in Escherichia coli. Pak J Biol Sci. 2010 May 15; 13(10), 2010. 504-508.
- LeThanh, H.; Neubauer, P.; Hoffmann, F. (2005): The small heat-shock proteins IbpA and IbpB reduce the stress load of recombinant Escherichia coli and delay degradation of inclusion bodies. Microb Cell Fact 4, 6; 2005.
- Martínez-Gómez A.I.; Martínez-Rodríguez S.; Clemente-Jiménez J.M.; Pozo-Dengra J.; Rodríguez-Vico F.; Las Heras-Vázquez F.J. (2007): Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids. Appl Environ Microbiol. 73(5); 2007. 1525-1531.
- Kotowska M.; Pawlik K.; Smulczyk-Krawczyszyn A.; Bartosz-Bechowski H.; Kuczek K. (2009): Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate. Appl Environ Microbiol. 75(4); 2009. 887-896.
Facts Worth Knowing
Escherichia Coli Bacteria
Escherichia coli is a bacteria type, that is non-spore-forming, Gram-negative and is characterized by its form of a straight rod. E.coli bacteria are present in the environment, foods, and intestines of humans and animals. E. coli is usually motile by using peritrichous flagella, but there are nonmotile types, too. E.coli are so-called facultatively anaerobic chemoorganotroph organisms, which means they are capable of both respiratory and fermentative metabolism. Most E.coli types are benign and fulfil useful functions in the body, e.g. suppressing the growth of harmful bacterial species, synthesising vitamins etc..
Escherichia coli bacteria cell of the so-called B type are a special category of E.coli strains, which are widely used in research to investigate mechanisms such as bacteriophage sensitivity or restriction-modification systems. Furthermore, E.coli bacteria are valued as reliable workhorse for protein expression in biotechnology and life science laboratories. For instance, E.coli are used to synthesize compounds such as proteins and oligosaccharides on industrial scale. Due to specific features such as protease deficiency, low acetate production at a high level of glucose, and enhanced permeability, E. coli B cells are he most frequently used host cells for the production of genetically engineered proteins.
Recombinant proteins (rProt) are are gaining significant importance in manifold branches, including in the chemical production, pharmaceutical, cosmetic, human and animal medicine, agriculture, food as well as waste treatment industries.
The production of recombinant protein requires the use of an expression system. As expressing cell systems for the production of recombinant DNA, both prokaryotic and eukaryotic cells can be used. Whilst bacterial cell are most widely used for protein expression due to factors such as low cost, easy scalability and simple media conditions, mammalian, yeast, algae, insect and cell-free systems are established alternatives. The protein type, functional activity, as well as the required yield of expressed protein influence the selection of the cell system used for protein expression.
In order to express recombinant protein, a particular cell must be transfected with a DNA vector containing the template of recombinant DNA. The cells transfected with the template are then cultured. As consequence of the cellular mechanism, the cells transcribe and translate the protein of interest, thereby producing the targeted protein.
As the expressed proteins are entrapped in the cellular matrix, the cell must be lysed (disrupted and broken) to release the proteins. In a subsequent purification step, the protein is separated and purified.
The first recombinant protein used in treatment was recombinant human insulin in 1982. Today, more than 170 types of recombinant protein are produced worldwide for medical treatments. Commonly used recombinant proteins used in medicine are for instance recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs, and enzymes for treating major diseases such as diabetes, dwarfism, myocardial infarction, congestive heart failure, cerebral apoplexy, multiple sclerosis, neutropenia, thrombocytopenia, anemia, hepatitis, rheumatoid arthritis, asthma, Crohn’s disease, and cancers therapies. (cf. Phuc V. Pham, in Omics Technologies and Bio-Engineering, 2018)