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Artificial non-coding RNA module for enhancing nitrogen fixation capacity of microorganisms

An artificial and nitrogen fixation technology, applied in the biological field, which can solve the problems of limited application and low nitrogen fixation efficiency.

Active Publication Date: 2020-11-13
北京绿氮生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The natural nitrogen fixation system is greatly affected by the environment, resulting in low nitrogen fixation efficiency, which greatly limits its application in agricultural production

Method used

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  • Artificial non-coding RNA module for enhancing nitrogen fixation capacity of microorganisms
  • Artificial non-coding RNA module for enhancing nitrogen fixation capacity of microorganisms
  • Artificial non-coding RNA module for enhancing nitrogen fixation capacity of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of AneR fusion expression vector

[0044] (1) Experimental method:

[0045]First, an artificial RNA module AneR with a full length of 505bp was synthesized by artificial chemical synthesis, and its expression was controlled by an artificial promoter element with a size of 360bp. Then, the artificial RNA module AneR and the expression vector pFLAα3 were subjected to Bam HI and Hind III double enzymes, respectively. Using T4 DNA ligase to insert the recovered AneR fragment into the multiple cloning site of pFLAα3, and finally verified by PCR sequencing, the AneR fusion expression vector pAneR was obtained. The expression vectors were transferred into three different nitrogen-fixing microbial chassis (Pseudomonas stutzeri, K. pneumoniae and A. vinelandii) respectively by means of triparental binding or electric shock , to obtain three recombinant nitrogen-fixing engineering strains.

[0046] (2) Experimental results:

[0047] The full-length nucle...

Embodiment 2

[0050] Expression Analysis of Artificial RNA Module AneR in Recombinant Engineering Bacteria under Example 2 Nitrogen Fixation Conditions

[0051] (1) Experimental method

[0052] 1. Activate the recombinant strains P.stutzeri(pAneR), K.pneumoniae(pAneR) and A.vinelandii(pAneR) in LB liquid medium, culture overnight at 30°C;

[0053] 2. Centrifuge the bacteria at 4000rpm / 10min the next day, and wash the bacteria twice with normal saline;

[0054] 3. Suspend the bacteria with physiological saline and adjust the OD 600 ≈1.0;

[0055] 4. Cultivate the bacteria under normal conditions and nitrogen fixation conditions respectively, and adjust the OD 600 ≈0.5;

[0056] 5. After shaking the culture solution at 30°C for 0.5h, centrifuge at 8000rpm for 5min to collect the bacteria;

[0057] 6. Use Promega large amount of RNA extraction kit Z3741 to extract bacterial total RNA, and perform single-stranded DNA (cDNA) inversion on the same amount of sample RNA used;

[0058] 7. The ...

Embodiment 3

[0063] The nitrogenase activity assay of embodiment 3 recombinant nitrogen-fixing engineering strains

[0064] (1) Experimental method

[0065] The determination of nitrogenase activity of recombinant engineering strains adopts the internationally recognized acetylene reduction method, and the specific steps are as follows:

[0066] 1. Activate the chassis strains P.stutzeri, K.pneumoniae and A.vinelandii and the recombinant strains P.stutzeri(pAneR), K.pneumoniae(pAneR) and A.vinelandii(pAneR) in LB liquid medium, overnight at 30°C to cultivate;

[0067] 2. Centrifuge the bacteria at 4000rpm / 10min the next day, and wash the bacteria twice with normal saline;

[0068] 3. Suspend the bacteria with physiological saline and adjust the OD 600 ≈1.0, the cells were cultured under the following conditions:

[0069] P.stutzeri and recombinant P.stutzeri(pAneR)

[0070] (1) Transfer the bacterial suspension to the ground Erlenmeyer flasks equipped with K medium (N-free), and adjus...

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Abstract

The invention relates to an artificial non-coding RNA module created by utilizing a synthetic biological method and application of the artificial non-coding RNA module in construction of an artificialnitrogen fixation system. The RNA module is interacted with a nitrogenase encoding gene nifHDK mRNA so that the stability of the nifHDK mRNA after transcription is enhanced, and the nitrogen fixationcapability of chassis microorganisms is further improved. A fusion expression vector of an artificial RNA module is constructed and transferred into different chassis nitrogen-fixing microorganisms.Experiments prove that the artificial RNA module can significantly improve the nitrogenase activity of the recombinant engineering strain under the nitrogen fixation condition.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to an artificial non-coding RNA module for enhancing the nitrogen fixation ability of microorganisms and its application in nitrogen fixation synthetic biology. Background technique: [0002] Biological nitrogen fixation is a physiological function unique to nitrogen-fixing microorganisms. This function is carried out under the catalysis of nitrogenase and is greatly affected by intracellular energy supply and environmental stress factors. To adapt to environmental changes, nitrogen-fixing microorganisms have formed a complex regulatory system during the evolution process. Nitrogen-fixing cells need to express and maintain sufficient nitrogen-fixing gene (nif) mRNAs to ensure efficient nitrogenase activity. [0003] The natural nitrogen fixation system is greatly affected by the environment, resulting in low nitrogen fixation efficiency, which greatly limits its application in agricult...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/78C12N15/74C12N1/21C12R1/38C12R1/22C12R1/065
CPCC12N15/113C12N15/78C12N15/74C12N9/0095C12Y118/06001C12N2330/30C12N2830/002C12N9/0004C12N2310/11C12N15/1137C12N2310/3519C12N2510/02
Inventor 林敏战嵛华燕永亮柯秀彬
Owner 北京绿氮生物科技有限公司