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Escherichia coli pressure response type promoter and preparation method thereof

A technology of Escherichia coli and promoters, applied in the field of bioengineering

Active Publication Date: 2018-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of high-intensity expression of foreign genes in Escherichia coli without adding inducers in different environments, the present invention provides an Escherichia coli stress-responsive promoter and a preparation method thereof

Method used

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  • Escherichia coli pressure response type promoter and preparation method thereof
  • Escherichia coli pressure response type promoter and preparation method thereof
  • Escherichia coli pressure response type promoter and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The construction of embodiment 1 promoter library ( figure 1 )

[0022] Construction of fluorescent protein GFP expression vector: construction of pCOLADet-GFP plasmid: use primers egfp F and egfp R as primers, and obtain the gene encoding GFP by PCR. The PCR product and plasmid pCOLADuet-1 were digested with BglII and XhoI respectively, and after recovery from the gel, they were ligated with T4 DNA ligase to construct the recombinant vector pCOLADet-GFP.

[0023] Construction of the promoter library: the sequence of the designed promoter library is as follows figure 1 shown, according to figure 1 Design primers as shown in Table 1 below, use pCOLADet-GFP as a template, and perform PCR with the primer pairs in Table 1 below to perform primer-mediated promoter replacement. The product amplified by PCR was digested with DpnI and directly transformed into BL21DE3, and spread on LB plate to obtain the promoter library.

[0024] Table 1

[0025]

[0026]

Embodiment 2

[0027] The screening artificial sequence of embodiment 2 promoter library

[0028] Transform the constructed recombinant plasmid containing the promoter into Escherichia coli BL21DE3, coat the plate to obtain transformants, pick the transformants to 96-well plate for culture (37°C, 200rpm), and measure the OD of the bacterial solution with a microplate reader 600 and GFP fluorescence intensity to screen mutant libraries with different expression levels. The recombinant Escherichia coli containing the promoter obtained through primary screening was fermented at the shake flask level to detect the GFP fluorescence intensity initiated by the promoter, which is re-screening, and the re-screening results are as follows: figure 2 shown.

Embodiment 3

[0029] Example 3 Test of the promoter's response to stress

[0030] The recombinant Escherichia coli containing the promoter obtained by re-screening was cultured under the different pressure conditions described in the following (1) to (4) (250mL Erlenmeyer flask, liquid volume 25mL, rotating speed 220rpm), and the fluorescence intensity of GFP was detected;

[0031] (1) Normal conditions: 37°C, pH ~ 7.0, LB medium;

[0032] (2) Low temperature: 20°C, pH ~ 7.0, LB medium;

[0033] (3) High osmotic pressure: Add final concentration of 0.5M NaCl to LB medium, 37°C, pH ~ 7.0, LB medium;

[0034] (4) Low pH: add citric acid buffer to the medium to adjust the pH to 4.5, 37°C, NaCl 0.17M, LB medium.

[0035] From image 3 It can be seen from A-3D that the screened promoters have stronger fluorescence intensity than the T7 promoter under different pressure conditions, especially the P21285 and 191 promoters.

[0036] The promoter nucleotide sequence is as follows:

[0037] Sequ...

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Abstract

The invention discloses an escherichia coli pressure response type promoter and a preparation method thereof and belongs to the field of promoter engineering. According to the escherichia coli pressure response type promoter, a pColaDuet-GFP plasmid is used as a template, and a degenerate primer is designed to connect conserved sequences of promoters identified by different sigma factors in series; intervening sequences are randomly mutated; green fluorescent protein GFP is used as a reporter gene to screen a plurality of promoters which have relatively high expression strength under low temperature, high osmotic pressure and low PH (Potential of Hydrogen) conditions.

Description

technical field [0001] The invention relates to an Escherichia coli pressure-responsive promoter and a preparation method thereof, belonging to the technical field of bioengineering. Background technique [0002] As a prokaryotic organism, Escherichia coli has many advantages of a prokaryotic expression system, such as clear genetic background, simple gene manipulation, short growth cycle, and high-density fermentation. At present, Escherichia coli has been widely used in basic research and large-scale industrial production, mainly in the expression of foreign proteins and the reconstruction of metabolic pathways to produce valuable target products. The application of a strong promoter in its expression system plays a key role in the high-efficiency expression of foreign proteins. [0003] In order to increase the yield of the target product, a common method in metabolic engineering is to use a strong promoter to overexpress the coding genes of key enzymes in the synthetic ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/70C12N1/21C12P21/00C12R1/19
CPCC12N15/102C12N15/113C12N15/70C12P21/00
Inventor 康振刘庆涛翁焕娇王阳陈坚堵国成
Owner JIANGNAN UNIV
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