Recombinant escherichia coli capable of efficiently decomposing formamide and phosphate oxide, and construction method and application thereof

A technology of recombining Escherichia coli and oxidizing phosphite, which is applied in the field of genetic engineering, can solve the problem of "killing" and achieve the effects of strong purpose, no contamination of bacteria products, and high material purity

Active Publication Date: 2018-03-06
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But microorganisms are everywhere, especially some microorganisms with spore structure, steam sterilization may not be able to "kill" them

Method used

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  • Recombinant escherichia coli capable of efficiently decomposing formamide and phosphate oxide, and construction method and application thereof
  • Recombinant escherichia coli capable of efficiently decomposing formamide and phosphate oxide, and construction method and application thereof
  • Recombinant escherichia coli capable of efficiently decomposing formamide and phosphate oxide, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Acquisition of formamidase gene for

[0055] Cultivate Paenibacillus pasadenensis.CS0611 with LB medium for one day at 37°C and 180rpm; collect the cultured cells by centrifuging at 4°C and 8000rpm for 5 minutes, and wash them twice with normal saline to remove residual culture base, and then extract the Paenibacillus paenibacillus pasadenensis.CS0611 genome according to the specific method of the OMEGA bacterial genome DNA extraction kit;

[0056] Using the extracted Paenibacillus pasadenensis.CS0611 genome as a template, A1(5'-CGC GATGAACGGACTGGGCGGCTTGAAC-3') and A2(5'-CCG CGTCGCGCCGCGCCTCCCTTCGCTC-3') are the upstream and downstream primers respectively, and the formamidase gene is amplified by PCR;

[0057] The enzyme reagent used in PCR is TaKaRa company's HS DNA Polymerase with GCbuffer; PCR reaction system and conditions are as follows:

[0058]

[0059]

[0060] PCR reaction conditions: react at 94°C for 5min; then react at 98°C for 10s, 55°C for 5...

Embodiment 2

[0063] Acquisition of phosphite dehydrogenase gene ptx

[0064] The phosphite dehydrogenase gene was derived from Klebsiella pneumonia.OU7, which was independently screened, and was obtained through independent screening. The screening medium used Monkina solid medium (g): glucose 10, (NH 4 ) 2 SO 4 0.5, MgSO 4 ·7H 2 O 0.3, NaCl 0.3, KCl 0.3, FeSO 4 ·7H 2 O 0.0036, MnSO4 ·H 2 O 0.03, KH 2 PO 3 0.3, Agar 20, dilute to 1L with ultrapure water;

[0065] Dip the single colony grown on the screening plate with an inoculation needle, and then continue to grade and streak on a new screening plate until a single colony appears to achieve purification, and finally isolate and purify a strain that grows on this screening medium A very good strain, through 16S rDNA sequencing, showed that it was Klebsiella pneumonia.OU7.

[0066] According to the specific method of the OMEGA Bacteria Genomic DNA Extraction Kit, the genome of Klebsiella pneumonia. -GA GATGCTGCCGAAACTCGTTATA...

Embodiment 3

[0072] Construction of Recombinant Bacteria BL21(DE3)(pETDuet-for)

[0073] The gene fragment for and plasmid pETDuet-1 obtained in Example 1 were double-digested with BamH I and EcoR I respectively. The underlined italics of the upstream primers were the BamH I enzyme cut points, and the underlined italics of the downstream primers were the EcoR I enzyme cut points. Cutting conditions: 37°C, enzyme digestion for 60 minutes;

[0074]

[0075] The digested products were recovered by gel cutting, and the recovery method and steps refer to the gel recovery kit of OMEGA Company; after recovery, 1wt% agarose gel electrophoresis was used to detect the recovery rate; then the recovered target fragment was connected to the plasmid, The ligation kit uses T4DNA Ligase from Thermo Fisher SCIENTIFIC, and the ligation system is as follows:

[0076]

[0077]

[0078] The molar ratio of target fragment to plasmid is 3:1.

[0079] Transform the ligation product into Escherichia col...

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Abstract

The invention discloses a recombinant escherichia coli capable of efficiently decomposing formamide and phosphate oxide, and a construction method and application thereof. The recombinant escherichiacoli takes escherichia coli DH5 alpha engineering bacteria as a host, formamidase gene and phosphorous acid dehydrogenase gene formed by cloning are sequentially connected with a vector to form recombinant plasmid, the recombinant plasmid is transferred into an escherichia coli expression strain (DE3), and the recombinant escherichia coli is formed through induction culture. The recombinant escherichia coli contains the formamidase gene and the phosphorous acid dehydrogenase gene at the same time, can express that formamidase decomposes formamide to produce NH4<+> and phosphorous acid dehydrogenase oxidizes phosphite into phosphate at the same time, and provides a nitrogen source and a phosphorus source required for growth and reproduction of the recombinant escherichia coli engineering bacteria, and other microorganisms do not have the two nutrition obtaining approaches, so that the recombinant escherichia coli becomes dominant bacteria, the problem of contamination of escherichia coli during industrial fermentation is solved, and meanwhile the demands of fermentation equipment is reduced and the additive volume of antibiotic is reduced.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Escherichia coli capable of efficiently decomposing formamide and oxidizing phosphite, and a construction method and application thereof. Background technique [0002] In recent years, with the rapid development of the fermentation industry, fermentation technology and fermentation equipment have been continuously updated. With more and more experience in fermentation management and more mature technical means, the fermentation contamination rate of various enterprises has also declined. However, bacteria contamination still occurs from time to time in the industrial fermentation process, and once the bacteria is contaminated, it will not only cause waste of raw materials, but also affect production efficiency, take a lot of time and effort, destroy production plans, disrupt production order, etc. Therefore, to solve the bacterial contamination in fermentation is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC12N9/0004C12N9/80C12N15/70C12Y120/01001
Inventor 娄文勇区晓阳李慧娴宗敏华徐培魏萍
Owner SOUTH CHINA UNIV OF TECH
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