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A method for improving acid stress resistance of recombinant Escherichia coli

A technology of Escherichia coli and acid stress, applied in the field of microbial engineering, to achieve the effect of improving acid stress resistance and simple operation

Active Publication Date: 2022-02-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the problem of the lack of a new method for improving the acid stress resistance of Escherichia coli with good effect, good genetic stability, simple operation, low cost and high success rate in the prior art, the present invention provides an acid stress resistant method. Recombinant escherichia coli with improved sex, said recombinant escherichia coli comprises a recombinant plasmid, said recombinant plasmid is an expression vector connected with a purpose gene; said purpose gene is the gene encoding trehalose 6-phosphate hydrolase TreC

Method used

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  • A method for improving acid stress resistance of recombinant Escherichia coli
  • A method for improving acid stress resistance of recombinant Escherichia coli
  • A method for improving acid stress resistance of recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant strain E.coli K12 MG1655 / pTrc99a-TreC

[0037] Specific steps are as follows:

[0038](1) Based on the treC gene sequence in the NCBI database (the TreC gene encoding trehalose 6-phosphate hydrolase, involved in the metabolic pathway of trehalose 6-phosphate, the process of regulating the hydrolysis of 6-phosphate in the trehalose system) is designed as SEQ Primers pTrc99a / TreC-F and pTrc99a / TreC-R shown in ID NO.2 and SEQ ID NO.3;

[0039] (2) designing the primer loop p-pTrc99a-F and the loop p-pTrc99a-R shown in SEQ ID NO.4 and SEQ ID NO.5 respectively;

[0040] (3) Using the genome of E.coli K12 MG1655 as a template, using p-pTrc99a / TreC-F, p-pTrc99a / TreC-R as primers to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification, and obtain the PCR product;

[0041] (4) Using the vector pTrc99a as a template, using loop p-pTrc99a-F and loop p-pTrc99a-R as primers to obtain a linearized long fragment of the vector by ...

Embodiment 2

[0045] Embodiment 2: the growth situation of recombinant bacterial strain and control bacterial strain under normal conditions

[0046] Specific steps are as follows:

[0047] (1) The bacterial strains E.coli K12 MG1655 / pTrc99a-TreC, E.coliK12MG1655 / pTrc99a-TreB obtained in Example 1 and the control bacterial strain E.coli K12 MG1655 / pTrc99a were respectively inoculated in LB liquid medium for activation, and placed Cultivate at 220rpm in a shaker at 37°C for 12h to obtain seed liquid;

[0048] (2) Transfer the seed solution obtained in the above step (1) to LB liquid medium with an inoculation amount of 2% (v / v), and place it in a shaker at 37°C at 220rpm for cultivation; sampling every 2 hours , measure the OD value under the 600nm wavelength, and draw the growth curve (the growth curve obtained by drawing is as follows: figure 1 ).

[0049] The result is as figure 1 As shown, through the analysis of the growth performance test, after 12 hours of cultivation, the growth ...

Embodiment 3

[0051] Example 3: Tolerance test of recombinant strain E.coli K12 MG1655 / pTrc99a-TreC under itaconic acid stress (pH 4.2)

[0052] Specific steps are as follows:

[0053] (1) The control strain E.coli K12 MG1655 / pTrc99a and the recombinant strain E.coliK12MG1655 / pTrc99a-TreC obtained in Example 1 were respectively inoculated in LB liquid medium for activation, and cultured in a shaker at 37°C at 220rpm for 12h , to obtain the seed solution;

[0054] (2) Transfer the seed solution obtained in the above (1) to fresh LB liquid medium with an inoculum amount of 2% (v / v), culture at 220rpm in a shaker at 37°C for 4.5h, and cultivate to logarithm In the mid-growth period, the OD600 at this time is 1.4-1.5, and the culture medium is obtained;

[0055] (3) Centrifuge the culture solution obtained in step (2) for 5 min at 6000 rpm, collect the thalline, wash the thalline obtained twice with 0.85% PBS buffer solution, and resuspend in an equal volume of fresh clothing In conic acid L...

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Abstract

The invention discloses a method for improving the acid stress resistance of recombinant Escherichia coli, which belongs to the technical field of microbial engineering. The present invention uses the gene of trehalose 6-phosphate hydrolase TreC as the target gene and Escherichia coli as the expression host to successfully construct a class of Escherichia coli engineering bacteria that can be widely used in the preparation of food, medicine, feed and chemicals; the Escherichia coli The itaconic acid stress resistance of Bacillus engineered bacteria has been significantly improved, up to 163.5 times higher than wild strains; the succinic acid stress resistance of Escherichia coli engineered bacteria has been significantly improved, up to 2.3 times higher than wild strains.

Description

technical field [0001] The invention relates to a method for improving the acid stress resistance of recombinant Escherichia coli, belonging to the technical field of microbial engineering. Background technique [0002] Escherichia coli is an important host bacteria in prokaryotes. These bacteria are widely distributed in nature and have rich species diversity. They are not only ideal materials for studying chemistry, genetics, molecular biology and genetic engineering, and have important academic value in theory, but also have application value in important fields closely related to human life, such as industry, agriculture, animal husbandry, food and medicine. extremely high. At present, a variety of high-value organic acid bio-fermentation methods have been successfully applied, and some of them have tried to use Escherichia coli as the host to express, but there is often the problem of acid stress. [0003] As a precursor substance with great potential, succinic acid ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/56C12P7/56C12P7/54C12P7/40C12R1/19
CPCC12N9/2402C12N15/70C12P7/56C12P7/54C12P7/40C12Y302/01093
Inventor 张娟杨谨华堵国成陈坚
Owner JIANGNAN UNIV
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