GTP cyclohydrolase I gene folE and application

A hydrolytic enzyme, pfed760-fole technology, applied in the fields of hydrolytic enzymes, applications, genetic engineering, etc., can solve problems such as functions to be determined, and achieve the effect of improving screening efficiency

Inactive Publication Date: 2020-06-05
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes two benefits - firstly, its use comes naturally because they were found on vegetables grown by farmers or other crops where there was no known harmful bacteria like Escherichia coli (BGE) inside them during production. Secondly, this new discovery allows us to study how certain specific factors affect the activity of these enzyme molecules involved in making hormones called gamma lactone. These findings help scientists better identify potential sources of health hazards associated with eaten raw materials such as BGA.

Problems solved by technology

This patented problem addressed in this patents relates to improving the efficiency at producing follicular acid without generating excessively harmful substances like nitrogen oxide gamma ray emitters. Previous attempts were made either by adding antimicrobium salts alone or combining them together into mixtures containing both folic acids and folacycliridones. These approaches resulted in reduced yields when folic Acid had low solubility in aqueous solution. To overcome these issues, we developed new techniques involving modifying specific proteins within cells to enhance their activities and reduce unwanted side actions associated with folic ester degradation systems.

Method used

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  • GTP cyclohydrolase I gene folE and application
  • GTP cyclohydrolase I gene folE and application
  • GTP cyclohydrolase I gene folE and application

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1: GTP cyclohydrolase I gene folE knockout homology arm clone

[0058] 1. PCR amplification of upstream and downstream homology arms

[0059] The genome of food-borne Lactobacillus plantarum YM4-3 was extracted using the Bacterial Genomic DNA Extraction Kit (Betaike Biotechnology Co., Ltd., China), and the specific operation was performed according to the kit instructions. The upstream 970bp sequence of the folE gene coding region is used as the upstream homology arm, and the downstream 1001bp sequence is used as the length of the downstream homology arm; the extracted genome is used as a template, and the primer pair is used

[0060] up-EF(5'-CCC AAGCTT GAACGGCGTAATGGTCAAC-3', the underline is the Spe I restriction site)+up-ER(5'-TTGAATTGACCACTGTAATTAATTATCAAATGGTGCGG-3') and

[0061] down-EF(5'-CCGCACCATTTGATAATTAATTACAGTGGTCAATTCAA-3')+down-ER(5'-GC ACTAGT ACACGGTCAACCGGCCTGG-3', the underline is the Hind III restriction site) to amplify the upstream ...

Embodiment 2

[0068] Embodiment 2: folE gene knockout vector construction

[0069] Use restriction endonucleases Spe I and Hind III to carry out synchronous digestion of the sequenced correct gene knockout fragment and the temperature-sensitive plasmid pFED760 respectively. The digestion system is: Spe I, 1 μL; Hind III, 1 μL; 1×H buffer , 2 μL; gene knockout fragment or pFED760, 10-16 μL; add sterilized deionized water to 20 μL, digest at 37°C for 4 hours; recover the digested product, according to the target gene:vector = 4:1-2:1 (molar ratio ) after sample addition, T4 DNA ligase was added and ligated at 16°C for 12-16 hours. The ligation product was introduced into Escherichia coli DH5α competent cells by the heat shock transformation method, and then spread on the erythromycin-LB solid plate. After culturing overnight at 28°C, extract the plasmid from 10 to 15 single colony cells, and use Spe I and Hind III enzyme digestion to obtain a positive plasmid, named pFED760-folE.

Embodiment 3

[0070] Embodiment 3: folE gene knockout strain construction

[0071] 1. The folE gene knockout vector is introduced into Lactobacillus plantarum competent cells

[0072] Lactobacillus plantarum competent cells were prepared according to the method reported by Fei Yongtao (2015, master's degree thesis of South China University of Technology). Add 10 μL gene knockout vector pFED760-folE to 90-100 μL Lactobacillus plantarum competent, mix gently, and transfer to pre-cooled electric shock cup after 5 min in ice bath, and conduct electric shock according to the parameters of 12.5kv / cm, 200Ω. After the electric shock is completed, quickly add 900 μL of fresh MRS culture solution to the electro-cup, blow and mix gently with a pipette tip, transfer the mixture to a 1.5 mL sterile centrifuge tube, and incubate at 28°C for 2.5-3 hours to recover the cells; After cultivation, the bacterial solution was centrifuged at 8,000-10,000 rpm for 3 minutes, 900 μL of the supernatant was discarde...

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Abstract

The invention belongs to the field of microbial genetic engineering, and discloses a GTP cyclohydrolase I gene folE and application. A nucleotide sequence of the GTP cyclohydrolase I gene folE is shown as SEQ ID NO:1. The gene is derived from food-borne lactobacillus plantarum, is safe and can be used in the field of later-stage food fermentation. A gene knockout strain screening method can be used for improving the screening efficiency of knockout strains. The key role of gene folE gene in folic acid synthesis is proved, and a certain theoretical basis is provided for the research and development of folic acid synthesis functional food. The GTP cyclohydrolase I gene folE and application analyze that the gene is derived from food-grade microorganisms and is safe; and the GTP cyclohydrolaseI gene folE and application analyze that the gene folE gene has nothing to do with cell morphology and strain growth except playing an important role in folic acid synthesis.

Description

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Claims

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Application Information

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Owner KUNMING UNIV OF SCI & TECH
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