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Method for transforming bacillus genome

A Bacillus and genome technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of the research and increase of the temperature-sensitive characteristics of the modified plasmid pBAV1K, and achieve low difficulty and adaptability of plasmid transformation. wide effect

Active Publication Date: 2017-05-31
CHENGDU MYTECH BIOTECH
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Problems solved by technology

However, the paper did not study the temperature-sensitive characteristics of the transformed plasmid pBAV1K. In our work, we found that the temperature-sensitive replication characteristics of pBAV1K-T5-GFP in Bacillus subtilis still exist, but the restricted culture temperature is increased from 37 °C of pKS1. up to 45°C

Method used

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  • Method for transforming bacillus genome
  • Method for transforming bacillus genome
  • Method for transforming bacillus genome

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Experimental program
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Embodiment 1

[0047] (1) Cut pBAV1K-T5-GFP plasmid with EcoR I and Apa I endonucleases, and perform homologous recombination with the synthesized MCS fragment to obtain plasmid pBAV1K.

[0048] The endonuclease used in this experiment and follow-up experiments was the rapid endonuclease from Thermo Company, and the gel recovery kit (DE-02011) from Chengdu Fuji Biotechnology Co., Ltd. was used for fragment recovery. The MCS fragment was synthesized by Jinweizhi Biotechnology Co., Ltd. Fragments and homologous recombination used Tiangen's EsayGeno rapid recombination cloning kit (VI201-02), the E. coli strain was top10, and the KCM method was used to prepare competent cells. DE-01001).

[0049] (2) Design primers to amplify the pBAV1K fragment with synonymous mutation to delete the Nde I restriction site, connect the fragments by homologous recombination cloning, and obtain a plasmid named pBTS.

[0050] The primers in this experiment and subsequent experiments were synthesized by Jinweizhi ...

Embodiment 2

[0052] The IC fragment was synthesized from the whole gene and inserted between XbaI and PstI of pBTS by homologous recombination to obtain the plasmid pBTS-IC. What is obtained here is the pBTS-IC plasmid in the preferred scheme; pBTS-FR-X can refer to the plasmid for transforming the xylAB gene below.

Embodiment 3

[0054] 1. Design primers to amplify the 493bp fragment xyl-F upstream of the xylose xylAB operon of the Z12 strain, and insert it between the EcoR I and BamHI sites of pBTS to obtain the plasmid pBTS-xyl-F; amplify the downstream 620bp fragment xyl-R , Insert between BamHI and Hind III of plasmid pBTS-xyl-F to obtain plasmid pBTS-xyl plasmid (knockout xylAB operon). The high-fidelity enzyme uses toyobo's KOD-Plus high-fidelity polymerase (KOD-201).

[0055] 2. Design primers to amplify the 693bp fragment upstream of the pgsBCA operon of the Z12 strain, insert it between the EcoR I and BamHI sites of pBTS, obtain a 558bp fragment downstream of the amplified plasmid pBTS-pgs-F, insert the BamHI and BamHI of the plasmid pBTS-pgs-F Between Hind III, plasmid pBTS-pgs was obtained. The zeo fragment was synthesized from the whole gene and inserted into the BamHI site of pBTS-pgs to obtain the plasmid pBTS-pgs-Z (the pgsBCA operon was knocked out, but the homologous recombination fra...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a method for transforming a bacillus genome. According to the method, adaptability is high (plasmid transformation difficulty is low, pBTS plasmids can be replicated in various gram-positive bacteria, and genomes of various bacteria can be transformed), the same strain can be transformed repeatedly (a recombinant plasmid has two homologous recombination fragments, homologous recombination happens twice in sequence, the strain is likely to return to be of a wild type after the second time of homologous recombination, or a transformed strain is obtained after the second time of homologous recombination, and no fragment affecting continuous transformation such as a resistance maker and a plasmid replication element is left ), and genes having great influences on bacterial growth can be transformed (cre recombinase and a second kind of resistance maker system are introduced, the transformed strain obtained through the second time of homologous recombination can be screened based on resistance, wild type strain with high viability can be killed, then cre recombinase is expressed, the second kind of resistance maker is cut off, and residual fragments do not affect further strain transformation).

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for transforming a bacillus genome. Background technique [0002] Protein expression technology is one of the core technologies of modern biology. Protein expression can not only be used in biological research, but also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently commonly used expression systems include Escherichia coli, yeast, insect cells and mammalian cells, etc., but each of them has obvious advantages and disadvantages. Escherichia coli expression system is the most well-studied, and there are many options. The most commonly used is Novagen's pET expression system, which uses phage T7 RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Under the optimal conditions, the target protein can reach more than 50% of the total protein of Escherichia co...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/74C12R1/125C12R1/10C12R1/07C12R1/46C12R1/01
Inventor 任钧唐旭曹镜雷蕾樊超柴进凯曹富明孙楠范佳
Owner CHENGDU MYTECH BIOTECH
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