Ultrasonic-mediated microbial genetic transformation method and application thereof

An ultrasonic and microbial technology, applied in the field of microbial genetic transformation, to achieve low-cost, high-throughput effects

Inactive Publication Date: 2010-11-03
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If this method is improved, whether it can be applied to Gram-pos

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  • Ultrasonic-mediated microbial genetic transformation method and application thereof
  • Ultrasonic-mediated microbial genetic transformation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 (Texas red-labeled dextran transformed thermophilic anaerobic Ethanolobacillus as an example)

[0048] 1) Bacterial culture

[0049] Inoculate thermophilic anaerobic ethanol bacterium into deoxygenated RCM medium (recipe: yeast extract 3g / L, beef extract 10g / L, peptone 10g / L, soluble starch 1g / L, glucose 5g / L, cysteine Amino acid hydrochloride 0.5g / L, NaCl 3g / L, NaAc 3g / L, agar 0.5g / L, resazurin 2mg / L, add distilled water to make up to a total volume of 1L, adjust the pH value to 6.8, pass N 2 Deoxygenation, 115 ℃ damp heat sterilization for 15 minutes. RCM solid medium preparation: agar 1.25%, others are the same as RCM medium), and the culture bottle is a small green bottle with a completely flat bottom. Anaerobic culture at 60°C, when the bacterial solution grows to the logarithmic phase, the cell concentration should be: 10 9 cells / mL. (Note: Different species have different anaerobic microbial culture medium formulations. RCM medium is only suitable f...

Embodiment 2

[0054] Embodiment 2 (taking plasmid DNA transformation thermophilic anaerobic ethanol bacterium as example)

[0055] 1) Bacterial culture

[0056] Inoculate thermophilic anaerobic ethanol bacterium into deoxygenated RCM medium (recipe: yeast extract 3g / L, beef extract 10g / L, peptone 10g / L, soluble starch 1g / L, glucose 5g / L, cysteine Amino acid hydrochloride 0.5g / L, NaCl 3g / L, NaAc 3g / L, agar 0.5g / L, resazurin 2mg / L, add distilled water to make up to a total volume of 1L, adjust the pH value to 6.8, pass N 2 Deoxygenation, 115 ℃ damp heat sterilization for 15 minutes. RCM solid medium preparation: agar 1.25%, others are the same as RCM medium), and the culture bottle is a small green bottle with a completely flat bottom. Anaerobic culture at 60°C, when the bacterial solution grows to the logarithmic phase, the cell concentration should be: 10 9cells / mL. (Note: Different species have different anaerobic microbial culture medium formulations. RCM medium is only suitable for t...

Embodiment 3

[0063] Application of the low-frequency ultrasonic transformation method provided by the present invention in cell fermentation and metabolic engineering (taking nanoparticle transformation of Acinetobacter sp. as an example)

[0064] 1) Cultivate Acinetobacter to the logarithmic growth phase in a 5L reactor;

[0065] 2) Adding gold nanoparticles into the reactor.

[0066] 3) Start the ultrasonic wave continuously or intermittently: 40kHZ, 10 seconds. At this time, the nanoparticles will be taken into the cell, and the subsequent intracellular metabolic regulation process will be initiated.

[0067] 4) Washing the cells with sterile physiological saline, and then observing the distribution of the gold nanoparticles in the Acinetobacter by using a transmission electron microscope. If the conversion is successful, the gold nanoparticles will form a granular shadow inside the cell (see figure 2 ).

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Abstract

The invention relates to a low-frequency ultrasonic-mediated method for transforming microbial, which comprises the following steps: (1) culturing bacteria to a logarithmic phase in a liquid culture medium; (2) adding a substrate to be transformed into a glass bottle containing the liquid culture medium in the step 1; (3) putting the glass bottle in the step 2 into the cavity of an ultrasonic instrument; (4) starting the ultrasonic instrument, wherein the frequency is 0.1-1000KHz, the power is 0.01-1000 Watts, non-probe type contact is adopted, the whole transformation process is carried out in the solution of a bioreactor, and ultrasonic waves are continuously adjustable or intermittent; and (5) recovering cells, transferring, and coating a panel. The invention can be used for operating a single cell and a single reactor, can realize high flux, has the advantages of simple operation and voluntary control, and can realize remote control.

Description

technical field [0001] The invention belongs to the field of microbial genetic transformation, and in particular relates to a transformation method mediated by low-frequency ultrasonic waves to transform biological macromolecules such as DNA, RNA, polysaccharide and protein into cells. [0002] The invention also relates to the application of a transformation method mediated by low-frequency ultrasonic waves in difficult-to-transform microorganisms (Gram-positive bacteria) and microorganism genetic metabolism engineering. Background technique [0003] The transformation of thermophilic, anaerobic, and Gram-positive bacteria has always been a headache for scientists, and there have been few successful reports in the world. The reason is that because thermophilic bacteria adapt to their special living environment, unlike normal temperature bacteria, their cell membranes are thick and have low permeability, and foreign nucleic acids must pass through specific DNA-binding protei...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N13/00C12R1/01C12R1/145
Inventor 宋厚辉林璐黄巍徐健
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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