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Bacillus gene scarless knockout/introduction plasmids, methods and kits

A bacillus, traceless knockout technology, applied in the field of genetic engineering, can solve the problems of low probability of gene knockout or knockin, low success rate, long cycle, etc., to achieve the effect of easy gene manipulation and plasmid transformation, and improve efficiency

Active Publication Date: 2018-11-09
WUHAN KANGFUDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of low gene knockout or knock-in probability, heavy screening identification and verification workload, low success rate, and long cycle in the prior art, the present invention provides a bacillus gene traceless knockout / introduction plasmid

Method used

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  • Bacillus gene scarless knockout/introduction plasmids, methods and kits
  • Bacillus gene scarless knockout/introduction plasmids, methods and kits
  • Bacillus gene scarless knockout/introduction plasmids, methods and kits

Examples

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Effect test

Embodiment 1

[0059] Example 1 Knockout plasmid pEBKS194-GFP+ containing I-SceI restriction site

[0060] As described in the content of the present invention, the knockout plasmid containing the I-SceI restriction site can have multiple combinations. This example uses one of the plasmid forms, pEBKS194-GFP+, to illustrate the construction process in detail. All primer sequences are shown in Table 1.

[0061] Construction of vector pWEBKS3

[0062] Using the expression plasmid pWEBK15 in the CN201410430501.3 patent (the antibiotic resistance gene of this plasmid is only the kanamycin resistance gene) as a template, the phosphorylated upstream and downstream primers F1 and R1 are used for full plasmid PCR amplification The amplified product was digested with restriction enzyme Dpn I overnight at 37°C and recovered, digested with Bgl II and ligated, transformed into Escherichia coli DH5α, and spread on a 50 μg / mL kanamycin resistant plate to screen positive The clones were sequenced with primer P1...

Embodiment 2

[0069] Example 2 Construction of temperature-sensitive plasmid pTISWts-RFP expressing I-Sce I enzyme

[0070] As described in the summary of the present invention, the temperature-sensitive helper plasmid expressing the I-SceI enzyme can have a variety of combinations. In this example, the construction process of one of the plasmid forms, pEBTWts-RFP, is described in detail. All primer sequences are shown in Table 1.

[0071] (1) Construction of vector pHYWVts

[0072] Using pHY300PLK vector (the plasmid contains antibiotic resistance genes including tetracycline resistance gene and ampicillin resistance) as a template, primers F8 and R8 are used to amplify the vector fragment; a synthetic temperature-sensitive replicon DNA fragment (SEQ ID NO. 16) As a template, use primers F9 and R9 to amplify the temperature-sensitive replicon WVts gene fragment. The amplified products of the gene fragment and vector fragment are digested with Dpn I enzyme overnight (37°C) and recovered, using th...

Embodiment 3

[0077] Example 3 Knockout of aprE gene in Bacillus subtilis CICC10073 strain

[0078] (1) Construction of knockout vector pKS194GFP-aprEUD

[0079] The CTAB (hexadecyltrimethylammonium bromide) method was used to extract the genomic DNA of Bacillus as a template for gene amplification (reference: Acta Microbiology, 2006, 46 (1): 7-12.); with subtilis spores Bacillus CICC10073 genomic DNA was used as a template, primers F14 and R14 were used as primers to amplify the upstream homology arm aprE-UP of aprE gene, and primers F15 and R15 were used as primers to amplify the downstream homology arm aprE-down of aprE gene; GFP+ plasmid was used as a template to amplify the vector fragment with primers F4 and R6. The amplified product of the vector fragment was digested with Dpn I overnight at 37°C and recovered. The operating instructions of the seamless cloning kit were used for the vector fragment and two gene fragments. After ligation, Escherichia coli DH5α was transformed and spread o...

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Abstract

The invention discloses a bacillus gene traceless knockout / knockin plasmid and method, and a kit. The genome of the bacillus gene traceless knockout / knockin plasmid contains only one kind of resistance gene marked with a positive selection marker, a replicon capable of being duplicated in escherichia coli, a thermo-sensitive type replicon capable of being duplicated in bacillus, at least one I-Scel enzyme cutting site and only one kind of chromogenic protein gene, wherein promoters in front of the resistance gene and the chromogenic protein gene are both constitutive promoters in bacillus. The invention further provides a helper plasmid used for improving the knockout / knockin efficiency of the bacillus gene traceless knockout / knockin plasmid, and resistance gene and chromogenic protein gene in the genome of the helper plasmid are different from those of the knockout / knockin plasmid. By the adoption of the two plasmids, one or more target DNA sequences in the genome of bacillus can be modified continuously or iteratively, and the plasmids can be applied to multiple fields including bacillus genetic modification, metabolic process researching, functional genome researching and industrial application researching.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular to the seamless knockout / into plasmid, method and kit of Bacillus genes. Background technique [0002] Bacillus (Bacillus) is a family of bacteria, refers to the bacillus or cocci that can form spores (endophytes), including Bacillus, Bacillus, Clostridium, Desulfurized Enterobacter, and Bacillus Sarcina And so on, they have strong resistance to harmful external factors and are widely distributed. Many Bacillus species have been recognized by the US FDA as GRAS (Generally Recognized as Safe) strains, which have important applications in the fields of industry, agriculture, food, and medicine. Each genus of Bacillus has its own biological characteristics. Through molecular genetic breeding, strains with specific functions can be selected and used in all aspects of industrial and agricultural production. Bacillus gene knock-out technology is an important method to study gene fu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75
CPCC12N15/75C12N2800/101C12N2820/10C12N2820/55
Inventor 汪小锋汪卫刘艳红张媛印容叶聪
Owner WUHAN KANGFUDE BIOTECH CO LTD
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