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Method for producing ice nuclei protein by recombinant escherichia coli fermentation

A technology for recombining Escherichia coli and ice nucleation protein, which is applied in the fields of genetic engineering and microbial fermentation, can solve the problems of difficulty in stable production, low activity and high expression of ice nucleation active protein, and achieves a short lag period, high activity and high vitality. Effect

Active Publication Date: 2022-05-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when using the conventional high-density fermented protein method to produce ice-nucleated active protein, the expression level of ice-nucleated active protein is too high, the activity is low, and it is difficult to produce stably.

Method used

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  • Method for producing ice nuclei protein by recombinant escherichia coli fermentation
  • Method for producing ice nuclei protein by recombinant escherichia coli fermentation
  • Method for producing ice nuclei protein by recombinant escherichia coli fermentation

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1 Utilizes the method for recombinant escherichia coli fermentation to produce ice nuclei protein

[0061] 1. Construction of recombinant Escherichia coli TDTC-inp001 producing ice nucleation protein

[0062] 1.1 Amplification of ice nucleoprotein gene inp

[0063] Using the genomic DNA of Pseudomonas syringae (Psudomonas syringae) TDTC-IN 13 as a template, PCR amplification was performed with the following primers:

[0064] inp-F: ctaacaggaggaattaaccatgaatctcgacaaggcgttg

[0065] inp-R: gagctcggatccccatcgatctactcgacctctatccagtc

[0066] The high-efficiency fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase from Vazyme Biotech Co., Ltd. was used for PCR amplification. The PCR amplification program was: 95°C for 3min; 30 cycles of 95°C for 15s, 58°C for 15s, and 72°C for 2min; 72°C for 5min. The obtained PCR product was subjected to electrophoresis and then gel-cut, and the gel recovery kit of Omega Company was used for gel recovery (operated accordi...

Embodiment 2

[0111] Embodiment 2 Utilizes the method for recombinant escherichia coli to ferment and produce ice nuclei protein

[0112] 1. The recombinant Escherichia coli TDTC-inp004 producing ice nucleation protein, its construction method is as follows:

[0113] 1.1 Amplification of ice nucleoprotein gene inp

[0114] Using the genomic DNA of Pseudomonas syringae (Psudomonas syringae) TDTC-IN 13 as a template, PCR amplification was performed with the following primers:

[0115] inp-F: ctttaagaaggagatataccatgaatctcgacaaggcgttg

[0116] inp-R: gcaagcttgtcgacctgcagctactcgacctctatccagtc

[0117] The high-efficiency fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase from Vazyme Biotech Co., Ltd. was used for PCR amplification. The PCR amplification program was: 95°C for 3min; 30 cycles of 95°C for 15s, 58°C for 15s, and 72°C for 2min; 72°C for 5min. The obtained PCR product was subjected to electrophoresis and then gel-cut, and the gel recovery kit of Omega Company was used for ge...

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Abstract

The invention provides a method for fermenting and producing ice nuclei protein by recombinant Escherichia coli. The recombinant Escherichia coli is constructed by introducing the ice nucleoprotein gene or the ice nucleoprotein gene expression cassette into Escherichia coli through plasmids or integrating it into the chromosome of Escherichia coli by means of genetic engineering, and the recombinant Escherichia coli does not contain antibiotics resistance gene. The invention overcomes the disadvantage that most of the existing ice nucleation active protein producing bacteria are plant conditional pathogenic bacteria and have potential harm to ecology. The ice nucleation active protein product produced by the fermentation strategy and method of the present invention has the advantages of high activity, simple operation, low cost and good repeatability of the production method, is harmless to the environment, has high production intensity, and is suitable for stable large-scale industrial production .

Description

technical field [0001] The invention relates to the technical field of genetic engineering and microbial fermentation, in particular to a method for producing ice nuclei protein by recombinant Escherichia coli fermentation. Background technique [0002] Ice-nucleation-active bacteria are a type of bacteria that can catalyze and induce water in plants to produce ice nuclei under low temperature conditions, thereby causing frost. Ice-nucleation-active bacteria are widely epiphytic on plant surfaces (especially leaves). Under normal circumstances, due to the presence of free water in the cells, plants do not freeze even at low temperatures of -7 to -8°C, resulting in supercooling; when ice-nucleating active bacteria exist, this microorganism acts as the strongest heterogeneous ice Nuclear factor, which induces the formation of ice crystals, makes the plant tissue lose its supercooling effect, and then causes frostbite damage to the host plant. Ice-nucleation-active bacteria a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/70C12N15/90C12P21/02C12R1/19
CPCC07K14/21C07K14/245C12N15/70C12N15/902C12N1/20C12N2310/20
Inventor 苏毅姜旭晏礼明
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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