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Enterococcus faecalis quorum sensing gene switch system constructed in escherichia coli as well as expression vector, engineering bacteria and application thereof

A technology of genetically engineered bacteria and Enterococcus faecalis is applied in the field of genetic engineering to achieve the effects of easy operation, simplified fermentation process and simple method

Active Publication Date: 2021-02-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, it has become a research hotspot to effectively interfere and inhibit the generation of specific signal peptide molecules secreted outside the cell, the accumulation process of signal peptide molecules, and the maximum concentration of signal peptide molecules. However, the quorum sensing system of Enterococcus faecalis can The application of the characteristics of detecting cell concentration to construct gene switches in common engineering bacteria such as Escherichia coli has not been reported yet

Method used

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  • Enterococcus faecalis quorum sensing gene switch system constructed in escherichia coli as well as expression vector, engineering bacteria and application thereof
  • Enterococcus faecalis quorum sensing gene switch system constructed in escherichia coli as well as expression vector, engineering bacteria and application thereof
  • Enterococcus faecalis quorum sensing gene switch system constructed in escherichia coli as well as expression vector, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of the main components of the gene switch system based on the quorum sensing system of Enterococcus faecalis

[0045] 1. Strains and plasmids

[0046] Enterococcus faecalis (Enterococcus faecalis) was purchased from Jiangsu Lvke Biological Company, Escherichia coli (E.colik12) strains and expression vectors pACYC Duet-1, pCDFDuet-1, pETDuet-1 were from Novagen (Darmstadt, Germany ), the PMD19T simple vector was purchased from Takara Company, and the dry powder of the expression vector containing the green fluorescent protein gene egfp was purchased from Nanjing Shuangling Biology.

[0047] 2. Enzymes and other reagents

[0048] Various antibiotics including ampicillin and chloramphenicol were purchased from Shanghai Sangong Co., Ltd. All chemical reagents were of analytical grade and purchased from Shanghai Sangong Co., Ltd. Various restriction endonucleases and DNA ligases were purchased from Thermo. 1kb DNALadder, plasmid mini-extraction kit, gel recovery ...

Embodiment 2

[0107] Construction of an expression vector based on the gene switch system of the quorum sensing system of Enterococcus faecalis

[0108] The construction of the quorum sensing system expression plasmid, the specific construction schematic diagram is as follows figure 1 shown.

[0109] The Pprgq promoter was connected to EcoNI and XhoI of double-digested pCDFDuet-1 through EcoNI and XhoI double digestion, and NcoI / BamHI and other restriction sites were added before XhoI to obtain pCDF-Pprgq (EcoNI / XhoI ) plasmid, the green fluorescent protein gene egfp was connected to the NcoI / BamHI of pCDF-Pprgq (EcoNI / XhoI) to obtain pCDF-Pprgq-egfp (ie C-Pprgq-egfp). The hybrid promoter trc (zu) of trp and lac is removed from the lacO operon, and the constitutive promoter trc (lost operon) that can constitutively initiate gene expression under any carbon source condition is obtained. The sequence is shown in SEQ ID NO: 14 As shown, the double-digested trc (lost operon) was connected to ...

Embodiment 3

[0113] Construction and fermentation of a fermentation strain that autonomously and dynamically regulates the expression of green fluorescent protein

[0114] Competent preparation of Escherichia coli

[0115] The transgenic recipient used in this example is Escherichia coli (Escherichia coli BL21), which comes from Novagen (Darmstadt, Germany). A single colony of Escherichia coli (Escherichia coli BL21) was picked in 5 mL of LB medium, and shaken Bed culture (37 ℃, 200rmp) overnight (about 12h). Take 500 μL of the overnight culture and transfer it to 50 mL of fresh LB medium, culture on a shaker (37°C, 200rmp) until the absorbance at 600nm reaches 0.35-0.6. 0.1mol / L CaCl 2 The solution was pre-cooled on ice. Pipette 25mL of bacterial liquid into a 50mL EP tube and place on ice to cool for 10min. Centrifuge (3000g, 5min, 4°C), discard the supernatant, add 1.666mL pre-cooled 0.1mol / L CaCl 2 Solution, use a pipette gun to gently suck up and down to mix well, so that the cel...

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Abstract

The invention discloses an enterococcus faecalis quorum sensing gene switch system constructed in escherichia coli as well as expression vector, engineering bacteria and application thereof. The enterococcus faecalis quorum sensing gene switch system comprises a heptapeptide pheromone cCF10 gene ccfa of enterococcus faecalis, a negative regulation pheromone peptide gene prgx and a promoter sequence Pprgq. The gene switch system provided by the invention is a gene switch system which does not depend on an inducer and can spontaneously and dynamically adjust target gene expression, fermentationengineering bacteria capable of starting target gene expression when OD600 reaches 1 are constructed by using the system. According to the inventioin, the starting strength is 10 times higher than that of the current commonly used vibrio fischer quorum sensing system. According to the invention, gene elements are respectively constructed on different expression vectors, and a gene switch system isconstructed in engineering bacteria, so that the engineering bacteria have the functions of spontaneously and dynamically regulating metabolic flow of fermentation strains and controlling gene expression, so that addition of an inducer can be avoided, the fermentation cost is saved and the fermentation process is simplified.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a quorum-sensing gene switch system for Enterococcus faecalis constructed in Escherichia coli, an expression vector, engineering bacteria and applications thereof. Background technique [0002] Fermentation engineering bacteria need to synthesize a large amount of recombinant protein and build a synthetic pathway. If a large amount of recombinant protein is expressed at the starting point of culture, it will compete with cell metabolism for intracellular resources, which will bring a huge burden to the bacteria. The imbalance of metabolic network will lead to some toxic intermediates. The accumulation of products will also cause toxicity to the growth of the bacteria, making the strain unable to maintain a good growth state and affecting the yield of the final product. Therefore, the predecessors have explored a variety of operating elements to achieve the induction...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/65C12N1/21C12R1/19
CPCC07K14/315C12N15/70C12N15/65Y02A50/30
Inventor 吴俊俊周朋包美娇董明盛
Owner NANJING AGRICULTURAL UNIVERSITY
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