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Electrotransformation method of Cytophaga hutchinsonii

A technology of Hastelloy fiber phagocytosis and electrotransformation, applied in the biological field, can solve the problems of low transformation efficiency, unstable repeatability, complicated operation, etc., and achieve the effect of improving transformation efficiency and reducing lethality

Inactive Publication Date: 2012-07-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the genetic transformation of Cellulophage harveyi often adopts the method of conjugative transformation to complete the introduction of exogenous genes. For example, MARK J. McBRIDE et al. obtained mutants through the method of Tn4351 transposition, but this method is complicated in operation and low in transformation efficiency. Low, unstable and poor reproducibility, so finding a new transformation method is a necessary prerequisite to complete the genetic manipulation of Cellulophage harveyi
[0005] The electroporation method is widely used in many microorganisms due to its advantages of simple operation and high transformation efficiency. However, for Cellulophage harveyi, it is difficult to complete electroporation due to its special culture conditions and the characteristics of the cell surface structure containing mucopolysaccharides. , there are few reports at home and abroad

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  • Electrotransformation method of Cytophaga hutchinsonii
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  • Electrotransformation method of Cytophaga hutchinsonii

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Embodiment 1

[0033] A method for electrotransformation of Cellophagus Hartneri, the steps are as follows:

[0034] (1) Extract plasmid pEP4351 (constructed by A J COOPER et al., see J. Bacteriol. 1997, 179(20): 6221) from E.coli S17-1λpir BW19851 strain with Takara Plasmid Purification Kit, and measure the plasmid concentration by spectrophotometer It is 40μg / ml recombinant plasmid solution;

[0035] (2) Preparation containing MgCl 2 and glycerol in water, MgCl 2 The final concentration of the solution was 8 mM, and the volume of glycerol accounted for 10% of the total volume of the solution, and the above aqueous solution was placed in an ice bath to prepare an electric shock buffer;

[0036] (3) Cultivate and activate the cells of cellophagocytosis in liquid medium TY2 at 170 rpm and 30 °C for 40 h, then transfer to liquid medium TY2 liquid medium, cultivate at 170 rpm, 30 ° C for 40 h, and then, at 7000 rpm , 5min, 4°C centrifugation to collect cells; wash and resuspend cells with pr...

Embodiment 2

[0046] Experiment on the effect of different electric field strengths on the electrotransformation efficiency of Cellophaga harvetii

[0047]In the experiment, the strain of Cellophagus harveta ATCC 33406 was used, and the plasmid pEP4351 was extracted from E.coli S17-1λpir BW19851 strain with Takara Plasmid Purification Kit, and the concentration of the plasmid was accurately determined by spectrophotometer to be 40 μg / ml. The shock buffer was formulated as follows: 10% glycerol, 8 mM MgCl 2 , chill on ice before use.

[0048] The logarithmic phase of Cellophaga harveii ATCC 33406 was inoculated into liquid medium TY2, cultured at 170 rpm, shaking at 30 °C for 40 h, 7000 rpm, 5 min, and centrifuged at 4 °C to collect cells; wash and resuspend cells with pre-cooled sterile water, Cell pellets were collected by centrifugation at 7000 rpm, 5 min, 4 °C, and the supernatant was discarded to remove impurities in the medium; the cells were washed and resuspended twice with pre-cool...

Embodiment 3

[0052] Different MgCl 2 Experiment on the effect of concentration on electrotransformation efficiency of Cellophaga harveta

[0053] In the experiment, the strain of Cellophagus harveta ATCC 33406 was used, and the plasmid pEP4351 was extracted from E.coli S17-1λpir BW19851 strain with Takara Plasmid Purification Kit, and the concentration of the plasmid was accurately determined by spectrophotometer to be 40 μg / ml.

[0054] The shock buffer is composed of different concentrations of MgCl 2 10% glycerol solution for the purpose of determining the optimal MgCl 2 concentration, shock buffer was stored frozen on ice.

[0055] The logarithmic phase of Cellophaga harveii ATCC 33406 was inoculated into liquid medium TY2, cultured at 170 rpm, shaking at 30 °C for 40 h, 7000 rpm, 5 min, and centrifuged at 4 °C to collect cells; wash and resuspend cells with pre-cooled sterile water, Cell pellets were collected by centrifugation at 7000 rpm, 5 min, 4 °C, and the supernatant was disc...

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Abstract

The invention relates to an electrotransformation method of Cytophaga hutchinsonii, and the method comprises the steps of: (1) preparing an exogenous DNA plasmid; (2) preparing a water solution containing MgCl2 and glycerin, and putting the water solution in an ice bath so as to obtain an electric shock buffer solution; (3) preparing a Cytophaga hutchinsonii thallus with a preservation number of ATCC NO.33406 into a competent cell; and (4) conducting electric shock, then transferring the competent cell into a liquid medium TY2 for recovery culture, and screening a transformant in a solid plate erythromycin-containing culture medium TY2, thus obtaining the electrotransformed Cytophaga hutchinsonii.

Description

technical field [0001] The invention relates to an electrotransformation method, in particular to an electrotransformation method of cytophaga harveta, and belongs to the field of biotechnology. Background technique [0002] Cytophaga hutchinsonii (Cytophaga hutchinsonii) is a kind of aerobic Gram-negative bacteria that can completely degrade crystalline cellulose. Since C. harveta doesn't contain cellosome-like structures like anaerobic cellulose-degrading bacteria, nor does it secrete free cellulase, its novel cellulose-degrading mechanism has so far remained elusive. Because it can completely degrade crystalline cellulose, it has potential application prospects in promoting the conversion and utilization of cellulose bioenergy. [0003] In view of its unique cellulose degradation mechanism and good application prospects, it is extremely important to carry out functional research at the molecular level, which is necessary to further explain its cellulose degradation mecha...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 卢雪梅李鹏伟徐元喜季晓飞张为灿
Owner SHANDONG UNIV
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