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Bacillus gene traceless knockout/knockin plasmid and method, and kit

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

Active Publication Date: 2016-04-20
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 traceless knockout/knockin plasmid and method, and kit
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  • Bacillus gene traceless knockout/knockin plasmid and method, and kit

Examples

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Effect test

Embodiment 1

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

[0060] As mentioned in the content of the present invention, the knockout plasmid containing the I-SceI restriction site can be in various combinations. This example will describe in detail the construction process of one of the plasmid forms, pEBKS194-GFP+. All primer sequences are listed in Table 1.

[0061] Construction of vector pWEBKS3

[0062] Using the expression plasmid pWEBK15 in the CN201410430501.3 patent (the only antibiotic resistance gene in this plasmid is the kanamycin resistance gene) as a template, use the phosphorylated upstream and downstream primers F1 and R1 to carry out PCR amplification of the whole plasmid , the amplified product was digested overnight at 37°C with restriction endonuclease DpnI, recovered, digested with BglII, ligated, transformed into Escherichia coli DH5α, spread on a 50 μg / mL kanamycin-resistant plate to screen positive clones, Sequencing with p...

Embodiment 2

[0069] Example 2 Construction of thermosensitive plasmid pTISWts-RFP expressing I-SceI enzyme

[0070]As mentioned in the content of the present invention, the temperature-sensitive helper plasmid expressing I-SceI enzyme can be in various combinations. This example will describe in detail the construction process of one of the plasmid forms, pEBTWts-RFP. All primer sequences are listed in Table 1.

[0071] (1) Construction of vector pHYWVts

[0072] Using the pHY300PLK vector (the antibiotic resistance gene of the plasmid has tetracycline resistance gene and ampicillin resistance) as a template, the vector fragment is amplified with primers F8 and R8; the synthetic thermosensitive replicon DNA fragment (SEQ ID NO.16) As a template, primers F9 and R9 were used to amplify the gene fragment of the temperature-sensitive replicon WVts. The amplified products of the gene fragment and the vector fragment were digested with DpnI enzyme overnight (37°C) and then recovered. The car...

Embodiment 3

[0077] Knockout of the aprE gene in the Bacillus subtilis CICC10073 bacterial strain of embodiment 3

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

[0079] Adopt CTAB (cetyltrimethylammonium bromide) method to extract the genomic DNA of bacillus as the template of gene amplification (reference literature: Acta Microbiology, 2006,46 (1): 7-12.); 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 the aprE gene, primers F15 and R15 were used as primers to amplify the downstream homology arm aprE-down of the aprE gene; pEBKS194- The GFP+ plasmid was used as a template, and the vector fragment was amplified with primers F4 and R6. The amplified product of the vector fragment was digested with DpnI enzyme at 37°C overnight and then recovered. Connect and transform Escherichia coli DH5α, spread on a 50 μg / mL kanamycin resistance plate to screen positive clones, use primers ...

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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 invention involves the field of genetic engineering, and specifically involves the genes of germioline genes without trace / pellets, methods and kits. Background technique [0002] Bacillus (Bacillus) is a family of bacteria, which refers to bacteria or bacteria that can form germii (endogenous cells), including Bacillus, genus of Bybulis, genus Rival, deoxye, and Bybulis Bacteria.Wait, they have strong resistance to the outside world and widely distributed.There are many genobacteriae species that are identified by the US FDA as Gras (GeneralReCognizeDassafe) strains, which have important application value in the fields of industry, agriculture, food and medical care.Each of Bacarbaccin has its own biological characteristics. Through molecular genetic breeding, it can be selected to nurture a specific functional bacteria, which is applied to all aspects of industrial and agricultural production.Bacillus gene knockout technology is an important means to ...

Claims

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Application Information

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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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