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Electro-transformation method for Listeria monocytogenes

A Listeria and electrotransformation technology, applied in microorganism-based methods, biochemical equipment and methods, and other methods of inserting foreign genetic materials, etc., which can solve problems that affect the use of competent cells, affect the progress of experiments, and cannot be stored for a long time. , to achieve the effect of increasing the electrical conversion efficiency, solving the preparation and conversion, and increasing the storage time

Inactive Publication Date: 2017-11-21
SHANGHAI ROYALTECH MED CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has great disadvantages in the process of using
First of all, due to the unsuitable control of the time of adding penicillin PNG, the subsequent listeria cell wall synthesis is quite different, and finally the competent efficiency of different batches of preparation varies greatly, which affects the use of competent cells.
Secondly, the competent state prepared by this method should be used within 30 minutes after the preparation is completed, and cannot be stored for a long time
Moreover, it takes a long time to prepare the competent state, which greatly affects the progress of the experiment.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0023] (1) Preparation of electroporation competent state

[0024] a) Overnight cultured Listeria was transferred to 200ml BHI medium at a ratio of 1:100, cultured with shaking at 37°C until OD 600 Value 0.2-0.25;

[0025] b) Add penicillin PNG at a final concentration of 10ug / ml, continue to cultivate for about two hours, until OD 600 to 0.5-0.6;

[0026] c) After 10 minutes of ice bathing, the cells were collected by centrifugation at 5000g for 10 minutes at 4°C;

[0027] d) resuspend the bacteria with 200ml 10% glycerol, and wash twice;

[0028] e) Resuspend the bacteria with 45ml of 10% glycerol, and add sterile lysozyme solution, the final concentration is 10ug / ml, bathe in water at 37°C for 20 minutes, invert and mix once every 10 minutes;

[0029] f) Collect the cells by centrifugation at 5000g for 10 minutes at 4°C, and wash the cells once with 20ml of 10% glycerol;

[0030] g) Resuspend the bacteria in 1ml of 10% glycerol, aliquot 100ul / tube, and store at -80°C. ...

Embodiment 1

5x 10 3

[0040] Table 1 The degree of protoplastization and electrotransformation efficiency of Listeria after treatment with different concentrations of lysozyme

[0041] Voltage (KV / cm)

Shock time (ms)

Transformation efficiency (unit / ug DNA)

8

6.3

8x 10 6

10

5.6

1x 10 7

12

4.9

4x 10 6

[0042] Table 2 Electrotransformation efficiency of Listeria under different voltages

[0043] Among them, in Table 1, the same amount of Listeria was treated with different concentrations of lysozyme to prepare electrotransformation competent cells, and the prepared competent cells were taken, diluted with hypertonic medium, and counted on a plate. The number of colonies with lysozyme concentration of 0 is recorded as N 0 , the number of colonies N grown after lysozyme treatment x , then the protoplast regeneration rate = (N x / N 0 ) x 100%. In addition, the prepared competent cells were serially diluted with sterile wa...

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PUM

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Abstract

The invention discloses an electro-transformation method for Listeria monocytogenes. The electro-transformation method is improved on the basis of original electro-transformation, owing to the fact that the effect of individual penicillin PNG is unstable, a small amount of lysozyme is added to be mildly treated, under the condition that bacterium bodies are not injured, the cell walls of gram-positive bacteria are further removed, and the electro-transformation efficiency is improved; and additionally, glycerinum containing saccharose serves as an electro-transformation medium, the bacterium bodies are better protected, and meanwhile the preservation time of minus 80 DEG C is increased. Through examining, the competence stored at minus 80 DEG C for half a year to a year can still have high transformation efficiency, and the difficult problems of preparation and transformation of the electro-transformation competence are solved.

Description

technical field [0001] The invention relates to a transformation method of exogenous plasmids, in particular to an electrotransformation method for Listeria. Background technique [0002] Listeria is widely distributed in nature and is an important foodborne pathogen. Immunocompromised people may be infected, and induce severe listeriosis, the mortality rate can reach 20-30%, so it has attracted great attention. Listeria is also an intracellular bacterium that can escape from cell lysosomes to replicate in the cytoplasm and express its own proteins, which can simultaneously induce inflammatory responses and activate the combination of MHC type I and type II antigen presentation pathways, making Listeria Bacteria has become a vaccine carrier with great application prospects. Today, some highly attenuated Lm mutant strains have been developed into vaccine candidate strains, and a series of vaccines targeting HPV, PSA, HER2 and other genes have been developed, some of which h...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12R1/01
CPCC12N15/90
Inventor 杨衡成秋香
Owner SHANGHAI ROYALTECH MED CO LTD
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