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A method for preparing microbial competent cells using atmospheric pressure room temperature plasma

A technology of competent cells and plasma, applied in the fields of biochemical equipment and methods, treatment of microorganisms and microorganisms with electricity/wave energy, etc.

Active Publication Date: 2016-02-24
WUXI TMAXTREE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no report about the preparation of microbial competent cells using atmospheric pressure and room temperature plasma

Method used

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  • A method for preparing microbial competent cells using atmospheric pressure room temperature plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of Escherichia coli Top10 strain competent cells and determination of transformation efficiency

[0037] 1. Materials and reagents

[0038] Material: recipient strain E.coliTOP10; plasmid: pUC

[0039] Reagents: LB liquid medium: peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0040] LB solid medium: agar 10g, peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0041] 2. Experimental equipment

[0042] ARTP (Atmospheric Room Temperature Plasma) Mutation Breeding System

[0043] 3. Experimental steps:

[0044] 1) Preparation of treatment solution: Pick a single colony from a solid plate growing Top10 strains and inoculate it in 5 mL of LB liquid medium, and incubate at 37°C for 12 to 16 hours; take 2 mL of the culture and inoculate it (5% inoculum) into 200 mL Continue to grow to OD in fresh LB liquid medium 600 0.5; t...

Embodiment 2

[0051] Example 2: Preparation of Escherichia coli DH5α Competent Cells and Measurement of Transformation Efficiency

[0052] 1. Materials and reagents

[0053] Materials: Recipient bacteria E.coliDH5α; plasmid pUC

[0054] Reagents: LB liquid medium: peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0055] LB solid medium: agar 10g, peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0056] 2. Experimental equipment

[0057] ARTP (Atmospheric Room Temperature Plasma) Mutation Breeding System

[0058] 3. Experimental steps

[0059] 1) Preparation of treatment solution: Pick a single colony on a solid plate growing DH5α strain and inoculate it in 5 mL of LB liquid medium, and incubate at 37°C for 12 to 16 hours; transfer 2 mL of the culture to inoculate (10% inoculum) into 200 mL Continue to grow to OD in fresh LB liquid medium 600 0.6; divide the bac...

Embodiment 3

[0066] Example 3: Preparation of Escherichia coli Top10 strain competent cells and determination of transformation efficiency

[0067] 1. Materials and reagents

[0068] Material: recipient strain E.coliTOP10; plasmid: pUC

[0069] Reagents: LB liquid medium: peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0070] LB solid medium: agar 10g, peptone 10g, yeast extract 5g, NaCl 10g, distilled water to volume 1L, pH 7.0, sterilized at 121°C for 20min.

[0071] 2. Experimental equipment

[0072] ARTP (Atmospheric Room Temperature Plasma) Mutation Breeding System

[0073] 4. Experimental steps:

[0074] 1) Preparation of treatment solution: Pick a single colony from a solid plate growing Top10 strains and inoculate it in 5 mL of LB liquid medium, and incubate at 37°C for 12 to 16 hours; take 2 mL of the culture and inoculate it (15% inoculum) into 200 mL Continue to grow to OD in fresh LB liquid medium 600 0.5 t...

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Abstract

The invention discloses a method for preparing microorganism competent cells by using constant-pressure room-temperature plasmas, and belongs to the application of a plasma technology in a biological technical field. According to the basic principle provided by the invention, energetic ions and charged particles in the plasmas are used for interacting with proteins and genetic materials in the microorganism cells, so as to finally cause the property change of the cells. Therefore, the cells are at a physiological status which is the most suitable for capturing and containing external DNA. The method provided by the invention uses the constant-pressure room-temperature plasmas with a liquid circulation system, and uses helium as a plasma discharging gas to take effect on the microorganism cells, so that the competent cells can be efficiently and conveniently prepared. The method is efficient, fast, safe and convenient, and can be widely used for as a novel experimental technique in fields including microbial genetics, molecular genetics and genetic engineering,.

Description

technical field [0001] The invention belongs to the application of plasma technology in the field of biology, and in particular relates to a method for preparing microbial competent cells by utilizing atmospheric pressure and room temperature plasma. Background technique [0002] The process of allowing DNA molecules with genetic information to enter host cells is called "transformation"; host cells that are easily accepted by foreign DNA molecules after physical or chemical treatment are called "competent cells". The state of competent cells directly affects the efficiency of transformation. In order to enable host cells to replicate or express specific DNA or proteins, obtaining competent cells that are easy to transform is the key. [0003] The methods for preparing competent cells mainly include physical methods and chemical methods. The more commonly used physical methods include electrical stimulation, ultrasound, osmotic pressure, etc.; chemical methods include CaCl ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N13/00
Inventor 毕鲜荣王立言
Owner WUXI TMAXTREE BIOTECHNOLOGY CO LTD
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