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Frequently asked questions about broken cells
Aug 10, 2018

1. E. coli expresses foreign protein. When sonicated, it is suspended in PBS containing 1% triton-X-100. The effect of ultrasound is better. The effect of 1% triton-X-100 is still obvious. Some other bacteria also work, such as Streptomyces. After expressing the recombinant protein, the cells were disrupted by ultrasonic cell disruption, using an ice bath, 400w, and crushed for 2s for 1s, but a large amount of foam was generated in a short time, which affected the crushing power. Both pbs and tris buffers were like this. Is the fragmentation incomplete, and the target protein is in these unbroken cells, what should I do?

Analysis: The bubble will be generated because the probe position is not placed well; the probe must be close to the bottom, about 0.5-1cm; the power will vary according to the instrument, but the liquid level can be observed, there is fluctuation but not too strong; you can choose ultrasonic cells The crusher is broken for 3s for 10s and broken for 20-30 times. The position of the horn should also be noted. If the sound is wrong, it should be adjusted in time; considering the concentration of bacteria. Try to increase the volume when crushing, the strength of the Zui should not exceed 60%; try to break 8s for 8s, for some bacterial proteins, it is difficult to dissipate the protein to cause protein denaturation to produce bubbles, which can be paused for a little longer. A protein found in the form of inclusion bodies.

2. What are the conditions for sonication of Streptomyces (actinomycetes)?

Analytical: actinomycetes belong to the (G+C) molar percentage (mol%) of the Gram-positive (Eubacteria) branching group on the phylogenetic tree of the prokaryotic system, which has the molecular and biological characteristics unique to prokaryotes. However, in its different groups, the chemical composition of the cell wall varies greatly. In the E. coli ultrasound, 400W is used, and the method of breaking 5s for 5s is effective, but it is used on Streptomyces, and it has no effect due to the difference in cell wall composition. The pre-spinning treatment of Streptomyces (actinomycetes) is generally configured to a certain concentration of bacterial suspension. The specific conditions for the crushing using the ultrasonic cell disruptor are: collecting the bacteria in the 24 h culture solution at 5 000 r / min for 5 min to collect the cells; washing with a pH 7.5 Na2HPO -NaH2PO buffer 3 times, and then The cells were formulated into a 1:3 bacterial suspension using the buffer. Placed in a 40 mL large plastic test tube; place the large plastic test tube in an ice bath and crush it with an ultrasonic cell disrupter (power 200W, 1/2" probe, crush for 30s, intermittent 30s); crushing solution at 12 000 r/min Centrifuge at high speed for 30 min, collect cell debris and supernatant. Wash cells can also be washed once with pre-cooled saline or Tris-HCl at pH 8. In addition, the ultrasonic dose varies greatly with the amount of sample and bacteria. The power can be 400-600w, crushed for 5s, stopped for 5s, ice bath, and the final concentration of 1 mM PMSF is added. In order to determine the appropriate ultrasonic intensity and frequency, it is necessary to check whether the bacteria are completely broken at any time.

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