Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Methylation protected host bacterium and construction method and application thereof

A technology of host bacteria and methylation, applied in the biological field, can solve the problems of non-methylation modification, low expression level, narrow application range, etc., and achieve the effect of normal expression

Pending Publication Date: 2019-11-12
莫纳(武汉)生物科技有限公司
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this method is that after obtaining methylation-protected host cells through cumbersome DNA library construction and screening, methylation-protected strains can only specifically protect DNA from cleavage by NdeI restriction enzymes, and few of them can be used. For the expression of other restriction enzymes, the scope of application is extremely narrow
Although the methylase M.CviPI has broad-spectrum protection, its specificity is weak, and it cannot methylate host cells against specific restriction endonucleases. Restriction endonucleases have certain effects on host cell DNA. Degradation, resulting in problems such as low expression and low protein yield

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Methylation protected host bacterium and construction method and application thereof
  • Methylation protected host bacterium and construction method and application thereof
  • Methylation protected host bacterium and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction of Methyltransferase M.Mae2481ORF83P Host Bacteria

[0077] (1) Obtain the methyltransferase M.Mae2481ORF83P gene

[0078] The PCR amplification method was used to obtain the methyltransferase M.Mae2481ORF83P gene from the genome of the bacterial strain Microcystis aeruginosaNIES-2481 using specific primers for the methyltransferase M.Mae2481ORF83P gene. The specific steps were as follows:

[0079] Plate-culture colony of strain Microcystis aeruginosaNIES-2481 resuspended in deionized water, incubated at 95°C for 10min, used as PCR template, nucleic acid sequence of SEQ ID NO:3-4 as PCR primer pair, using MonHI-FI DNAPolymerase, according to the standard procedure The PCR reaction obtained the M.Mae2481ORF83P gene fragment which was consistent with the theoretical value.

[0080] (2) Construction of methyltransferase expression vector

[0081] pACYC184 plasmid using FlashCut TM NdeI and FlashCut TM After XhoI double digestion, with MonClone ...

Embodiment 2

[0087] The construction of embodiment 2 restriction endonuclease NdeI recombinant expression strains

[0088] (1) Obtain restriction endonuclease NdeI gene

[0089] Using the method of PCR amplification, use the specific primer of restriction endonuclease NdeI gene from the genome of bacterial strain Neisseria denitrificans, obtain restriction endonuclease NdeI gene, specific steps are:

[0090] Plate-cultured colonies of the strain Neisseria denitrificans resuspended in deionized water, incubated at 95°C for 10 minutes, used as a PCR template, the nucleic acid sequence of SEQ ID NO:5-6 as a PCR primer pair, using MonHI-FI DNA Polymerase, and performing PCR according to the standard procedure The reaction obtained the R.NdeI gene fragment consistent with the theoretical value.

[0091] (2) Construction of restriction endonuclease NdeI expression vector

[0092] pBAD plasmid using FlashCut TM NdeI and FlashCut TM After HindIII double enzyme digestion, dephosphorylate with M...

Embodiment 3

[0098] The activity detection of embodiment 3 restriction endonuclease NdeI

[0099] The enzymatic activity of restriction endonuclease NdeI is defined as: 1 μL of restriction endonuclease NdeI can completely digest 1 μg of λDNA within 15 minutes in a 20 μL reaction system at 37°C.

[0100] In this example, the purified restriction endonuclease NdeI was serially diluted in 1×FlashOne TM Buffer (Mona Biotechnology Co., Ltd.) buffer conditions, in a 20 μL reaction system, incubated with the substrate λDNA at 37° C. for 60 min, and then detected the digestion of the substrate by 1% agarose gel electrophoresis. The result is as Figure 4 As shown, the purified restriction enzyme NdeI has good enzyme cutting activity, and the specific activity is about 900,000 U / mg induced bacterial liquid.

[0101] In summary, the present invention transforms the M.Mae2481ORF83P methyltransferase expression vector into the host bacteria to construct a methylation-protected bacterial strain, whi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a methylation protected host bacterium and a construction method and an application thereof. An amino acid sequence of a transmethylase is shown in SEQ ID NO:1 and a nucleotide sequence of the transmethylase is shown in SEQ ID NO:2, the host strain is transformed with a methyltransferase expression vector which realizes a methylation protection function of host bacterium genomic DNA and avoids a cleavage effect of NdeI on host DNA, and the methylation protected host bacterium can be used for expressing a variety of restriction endonucleases including NdeI.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a host bacterium and its construction method and application, in particular to a methylation-protected host bacterium expressing restriction endonuclease NdeI, its construction method and application. Background technique [0002] In organisms, there is a class of enzymes that can cut foreign DNA, limit the invasion of heterologous DNA and make it lose its vitality, but have no damage to its own DNA, thus protecting the original genetic information of cells. Because this cutting effect is carried out inside the DNA molecule, it is named restriction endonuclease (restriction enzyme for short). The discovery of restriction enzymes promoted the birth of DNA recombination technology, greatly promoted the development of modern molecular biology and genetic engineering, and is an indispensable basic tool for contemporary genetic engineering research. [0003] There is a "restriction...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/10C12N15/70C12N1/21C12N9/16C12R1/19
CPCC12N9/1007C12N9/16C12N15/70
Inventor 程槐旭刘杨燕东平聂尚海
Owner 莫纳(武汉)生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com