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A method for transforming Bacillus siamese by electric shock

A technology of Bacillus and electric shock transformation, applied in the field of microbial genetic engineering, can solve the problems of low transformation efficiency, no reports, and low cell membrane permeability, and achieve the effects of high transformation rate, stable effect and simple operation.

Active Publication Date: 2020-12-11
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0004] Since Bacillus is a Gram-positive bacterium with a thicker cell wall, the difficulty and low transformation efficiency of Bacillus have always been concerned.
At present, although there have been successful precedents for the genetic transformation method of Bacillus, except for the relatively mature transformation system of Bacillus subtilis model bacteria, the genetic transformation system of other wild-type Bacillus is still being tried and improved. There is no report on the method of bacillus genetic transformation or its genetic engineering
In addition, unlike model strains such as Bacillus subtilis 168, WB600 and WB800, wild-type Bacillus siamese usually has a strict restriction modification system, which is easy to degrade exogenous DNA, and its cell membrane permeability is generally low, which is not conducive to cell regeneration. The smooth entry and exit of internal and external substances, thus further increasing the difficulty of DNA transformation

Method used

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  • A method for transforming Bacillus siamese by electric shock
  • A method for transforming Bacillus siamese by electric shock
  • A method for transforming Bacillus siamese by electric shock

Examples

Experimental program
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Effect test

Embodiment 1

[0118] 1. Instrument or equipment:

[0119] pH meter PB-10 was purchased from Sartorius; biochemical incubator SPX-158L was purchased from Ningbo Xinzhi, China; Eppendorf centrifuge 5418 was purchased from Eppendorf; steam sterilizer YXQ50 was purchased from Xi’an Taikang; ultra-clean workbench SKJH -1109 was purchased from Shanghai Sukun; double-beam UV-visible spectrophotometer UV2310II was purchased from Hangzhou Aosheng; Nano Drop spectrophotometer ND-1000 was purchased from Nano Drop Company; electric shock conversion instrument MicroPulser was purchased from Bio-Red, USA.

[0120] 2. Strains, plasmids and primers:

[0121] Bacillus siamese JFL15 is isolated from fish intestines and has strong antibacterial activity against a variety of pathogenic bacteria and fungi. It is preserved in the Institute of Food Biotechnology, School of Food Science, South China Agricultural University, and has undergone whole genome sequencing (Genbank accession number: NZ_KQ236689 .1).

[...

Embodiment 2

[0156] The difference between this example and Example 1 is that the shuttle vector used is the pHT08-cody-up-down homologous recombination plasmid, and the rest are the same as Example 1.

[0157] The successfully constructed pHT08-cody-up-down plasmid was transformed into Bacillus siamese JFL15 by electroporation, and the results were as follows: Figure 7 As shown, a large number of transformants grew on the LB resistance plate containing 5 μg / mL chloramphenicol, indicating that the plasmid pHT08-cody-up-down may have successfully transformed Bacillus siamese JFL15, and the plasmid pHT08-cody-up- The transformation efficiency of down was significantly higher than that of plasmid pHT08-cas9-gRNA. Randomly pick 3 transformants in LB liquid medium containing 5 μg / mL chloramphenicol resistance, after overnight culture, extract the plasmid, the results are as follows Figure 8 As shown, the plasmid band is between 8000 and 15000 bp, which is consistent with the expected size of...

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Abstract

The invention discloses a method for transforming Bacillus siamensis by electric shock, belonging to the technical field of microbial genetic engineering. The invention comprehensively optimizes factors such as preparation of competent cell, concentration of plasmid, electric shock parameter and the like during electric shock transformation of Bacillus siamensis JFL15, and establishes a simple andeffective genetic transformation method of Bacillus siamensis for the first time. The genetic transformation method of Bacillus siamensis of the invention is simple in operation, high in conversion rate, stable in effect, and can be used for the knockout of CRISPR -cas9 gene and the large plasmid () 11kbp for homologous recombination knockout vector with high efficiency, not only laid a good foundation for genetic modification of Bacillus siamensis, and has an important significance for accelerating its application in industrial production, but also provided an important reference for genetictransformation of other Bacillus spp.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a method for transforming bacillus siamese by electric shock. Background technique [0002] Bacillus is an important class of biocontrol microbial resources, which can produce functional molecules such as metabolites and enzymes with various biological functions, so it is widely used in agricultural and aquaculture biological control, pharmaceutical development, industrial enzyme preparations, feed processing and environmental protection. pollution control and many other fields. Bacillus siamensis is a new type of Bacillus species, the secondary metabolites produced by it are effective against many pathogenic bacteria (such as Escherichia coli, Salmonella, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa bacterium, Vibrio parahaemolyticus and Vibrio vulnificus, etc.) and fungi (such as Fusarium oxysporum, Phytophthora capsici,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75
CPCC12N15/75
Inventor 林俊芳林龙镇郭丽琼许本宏
Owner SOUTH CHINA AGRI UNIV
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