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Functional microbial transformation method with combination of permeabilization and ultrasound

A functional microorganism and microbial technology, applied in the field of genetic engineering and cell engineering, can solve the problem of low transformation efficiency

Inactive Publication Date: 2013-05-08
宋一之
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for anaerobic Gram-negative bacteria, there is no corresponding ultrasonic transformation method put into practice. More importantly, although simple ultrasonic transformation can increase the transformation efficiency to 10 -6 order of magnitude, but with commercial electroconversion 10 -5 The conversion efficiencies above are still relatively low

Method used

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  • Functional microbial transformation method with combination of permeabilization and ultrasound
  • Functional microbial transformation method with combination of permeabilization and ultrasound
  • Functional microbial transformation method with combination of permeabilization and ultrasound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: The method for transforming Desulfovibrio vulgaris Hildenborough by permeabilization-ultrasonic transformation technology:

[0031] Unless otherwise specified, all operations were performed under sterile anaerobic conditions.

[0032] 1) Cell culture:

[0033] Desulfovibrio vulgaris Hildenborough (hereinafter referred to as DvH) is an anaerobic Gram-negative bacterium, which can be obtained from the German Microorganism Collection and Cell Culture Center (DSMZ, strain number DSM 644), and the culture medium is Medium 63 ( For the preparation method, refer to the DSMZ website instructions). Among the existing transformation techniques for DvH bacteria, only electroporation has been reported to successfully transform DNA into this bacteria.

[0034] Bacteria were inoculated into 50mL Medium63 medium and cultured at 30-35°C for 72h.

[0035] 2) Cell washing

[0036] Centrifuge the cultured bacterial solution at 4000rpm for 10min to collect the bacterial cell...

Embodiment 2

[0058] Example 2: The method of permeabilization-ultrasonic transformation technology for transforming Pseudomonas putidaKT2440:

[0059] Pseudomonas putida KT2440 (hereinafter referred to as KT2440) is an aerobic Gram-negative bacterium, which is available to the public through the German Collection of Microorganisms and Cell Culture (DSMZ, strain number DSM 6125). The culture medium adopts LB medium. Researchers have shown that DNA conversion to KT2440 can be carried out using ultrasonic transformation, and the conversion efficiency can reach up to 9.8×10 -6 . This example will show that the conversion efficiency can be greatly improved through the permeabilization-ultrasonic conversion technology.

[0060] 1) Cell culture:

[0061] Bacteria were inoculated into 5 mL of LB medium and cultured at 30 °C for 16 h.

[0062] 2) Cell washing

[0063] The bacterial solution was centrifuged at 4000rpm for 10min, the bacterial cells were collected, and washed twice with 80mmol / L...

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Abstract

The invention discloses a functional microbial transformation method with combination of permeabilization and ultrasound. The method comprises the following steps of: permeabilizing microbial cells by a Decon solution to reduce the content of phospholipids in cell walls and cytoplasmic membranes; adding calcium ions to a system during the permeabilization so as to facilitate the sedimentation and the transportation of exogenous DNA (Deoxyribonucleic Acid); and then exerting low-frequency ultrasonic waves of 20-80 kHz to transport the DNA and ribonucleic acid (RNA) to be transformed into the cells. By virtue of the Decon permeabilization, the power of the ultrasound in the ultrasonic transformation can be relatively reduced, the ultrasound time is short, and the damage of the ultrasound to the cells is alleviated; by reasonably choosing a permeabilization reagent and determining corresponding concentration, a medium solution simultaneously meeting the requirements of the permeabilization and the ultrasonic transformation is obtained; and exogenous nucleic acid molecule transformation of host bacteria in a larger range can be realized, and the transformation efficiency is enhanced, so that an effective technological way for genetic engineering transformation and new functional microbial culture is provided, the efficiency is enhanced, and the cost and the operation difficulty are reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and cell engineering, and in particular relates to a method for transforming functional microorganisms through the combination of permeabilization and ultrasound. Background technique [0002] The process by which bacteria acquire new genetic traits by absorbing foreign DNA is called transformation. The introduction of exogenous DNA molecules such as plasmids or double-stranded DNA into bacterial cells is one of the most basic technical means in microbiology and molecular biology, and is also one of the key processes in genetic engineering of microorganisms. After decades of research, there are relatively efficient DNA transformation methods for some model microbial systems. For example, for the Escherichia coli system, the transformation efficiency can reach 10 by heat stress after preparing competent cells. -5 or 10 -6 . For non-specific microbial hosts, more widely applicable tec...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 宋一之
Owner 宋一之
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