An artificial noncoding RNA module that enhances microbial nitrogen fixation

An artificial nitrogen fixation technology, applied in the biological field, can solve the problems of low nitrogen fixation efficiency and limited application, and achieve the effect of enhancing nitrogen fixation ability and nitrogenase activity

Active Publication Date: 2022-06-28
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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  • An artificial noncoding RNA module that enhances microbial nitrogen fixation
  • An artificial noncoding RNA module that enhances microbial nitrogen fixation
  • An artificial noncoding RNA module that enhances microbial nitrogen fixation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of AneR fusion expression vector

[0044] (1) Experimental method:

[0045]Firstly, an artificial RNA module AneR with a full length of 505 bp was synthesized by artificial chemical synthesis, and its expression was controlled by an artificial promoter element with a size of 360 bp. Then the artificial RNA module AneR and the expression vector pFLAα3 were subjected to Bam HI and Hind III double enzymes respectively. Then, T4 DNA ligase was used to insert the recovered AneR fragment into the multi-cloning site of pFLAα3. Finally, the AneR fusion expression vector pAneR was obtained by PCR sequencing verification. The expression vectors were transferred into three different nitrogen-fixing microbial chassis (P. stutzeri, K. pneumoniae, and A. vinelandii) by tri-philic binding or electrical stimulation. , and obtained three recombinant nitrogen-fixing engineering strains.

[0046] (2) Experimental results:

[0047] The full-length nucleic acid seq...

Embodiment 2

[0050] Example 2 Expression analysis of artificial RNA module AneR in recombinant engineering strains under nitrogen fixation conditions

[0051] (1) Experimental method

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

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

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

[0055] 4. The bacteria were cultured under normal conditions and nitrogen-fixing conditions, respectively, and the OD was adjusted. 600 ≈0.5;

[0056] 5. After the culture medium was shaken and cultured at 30°C for 0.5h, the cells were collected by centrifugation at 8000rpm for 5min;

[0057] 6. Use Promega Mass Extraction Kit Z3741 to extract total bacterial RNA, and reverse the single-stranded DNA (cDNA) with the same amount of sample RNA;

[0058] ...

Embodiment 3

[0063] Embodiment 3 The nitrogenase activity assay of recombinant nitrogen-fixing engineering strain

[0064] (1) Experimental method

[0065] The nitrogenase activity of the recombinant engineering strain was determined by the internationally recognized acetylene reduction method. The specific steps are as follows:

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

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

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

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

[0070] (1) Transfer the bacterial suspension to a grinding-mouthed conical flask equipped with K medium (without N), and adjust...

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Abstract

The invention relates to an artificial non-coding RNA module created by a synthetic biology method and its application in the construction of an artificial nitrogen fixation system. The RNA module enhances the post-transcriptional stability of the nifHDK mRNA by interacting with the nitrogenase encoding gene nifHDK mRNA, thereby improving the nitrogen fixation ability of the chassis microorganisms. The invention constructs the fusion expression vector of the artificial RNA module, and transfers it into different chassis nitrogen-fixing microorganisms. Experiments have confirmed that under nitrogen fixation conditions, the artificial RNA module of the present invention can significantly improve the nitrogenase activity of recombinant engineered strains.

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-fixing ability of microorganisms and its application in nitrogen-fixing synthetic biology. Background technique: [0002] Biological nitrogen fixation is a unique physiological function of 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. In order to adapt to environmental changes, nitrogen-fixing microorganisms have formed a complex regulatory system during evolution. 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 agricultural pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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