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Bacillus gene knockdown carrier plasmid pBD1 based on dCas9, construction and application

A technology of bacillus and vector plasmids, applied in the field of molecular biology and biology, to achieve the effect of simple construction of vectors and simple operation

Active Publication Date: 2016-07-20
NANYANG NORMAL UNIV
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  • Abstract
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Problems solved by technology

But knockdown efficiency is often a weakness of RNAi

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  • Bacillus gene knockdown carrier plasmid pBD1 based on dCas9, construction and application
  • Bacillus gene knockdown carrier plasmid pBD1 based on dCas9, construction and application
  • Bacillus gene knockdown carrier plasmid pBD1 based on dCas9, construction and application

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Embodiment Construction

[0047] The utility model will be further described below in conjunction with the accompanying drawings. It should be noted that the present embodiment is based on the technical solution and provides detailed implementation and specific operation process, but the protection scope of the utility model does not limited to this example.

[0048] Such as Figure 1-8 Shown, a dCas9-based bacillus gene knockdown vector plasmid pBD1 construction method comprises the following steps:

[0049] Construction of S1Pveg-SapI-sgRNA module

[0050] Primers Pveg-F-XbaI and Pveg-R were designed, and the plasmid psb1c3-pveg-RBS-dCas9-b0015 was used as a template to amplify the promoter Pveg constitutively expressed in Bacillus; the plasmid psb1c3-pveg-RBS-dCas9-b0015 The nucleotide sequence of the primer is shown in SEQIDNO: 1, and the primers Pveg-F-XbaI and Pveg-R sequences are as follows:

[0051] Primers Pveg-F-XbaI and Pveg-R were designed, and the plasmid pPDB constructed by Sun Baolin'...

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Abstract

The invention discloses a bacillus gene knockdown carrier plasmid pBD1 based on dCas9, construction and application. The plasmid pBD1 is constructed by utilizing multiple molecular biology methods such as PCR, enzyme digestion, enzyme linking, sequencing and transformation, the plasmid pHY300-dCas9-sgRNA (pBD1) containing IPTG an inducible Pgrac promoter, dCas9 and sgRNA is constructed on the basis of bacillus expression vector, and the plasmid is theoretically suitable for a broad bacillus range because of having a large promoter strength and a wide host range.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a construction method and application of a dCas9-based bacillus gene knockdown vector plasmid pBD1. Background technique [0002] With the maturity of molecular cloning technology, the functional research of some important genes relies on gene knockdown technology. Gene knockdown is a reverse genetics research method. Through a certain way, a specific gene in the body is deleted or inactivated or the expression level is reduced from the molecular level, and the changes in the apparent traits are compared to understand the function of the gene. Methods have extremely important theoretical and practical significance in many research fields such as medicine and biology. For the first time, a complete embryonic stem cell gene knockout mouse model was established based on homologous recombination, and then homologous recombination became the main way to knock down ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/75
CPCC12N15/66C12N15/75
Inventor 张林牛秋红龚姝榕符强惠丰立阚云超
Owner NANYANG NORMAL UNIV
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