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Construction and application of cysteine single-cell biosensor

A biosensor, cysteine ​​technology, applied in microorganism-based methods, microbial determination/inspection, microorganisms, etc., can solve the problem of lack of rapid, sensitive and highly specific detection, achieve real-time detection, enhance downstream genes Expression, strong specificity

Inactive Publication Date: 2019-09-27
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the current lack of rapid, sensitive and highly specific methods for detecting L-cysteine, the present invention provides a single-cell L-cysteine ​​biosensor based on the transcriptional regulator CcdR and its construction method. Biosensor can convert L-cysteine ​​concentration into fluorescence intensity signal with high sensitivity

Method used

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  • Construction and application of cysteine single-cell biosensor
  • Construction and application of cysteine single-cell biosensor
  • Construction and application of cysteine single-cell biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of L-cysteine ​​biosensor expression plasmid

[0031] In this example, PCR amplification is performed on each component module of the L-cysteine ​​biosensor to obtain the nucleotide fragments that make up the module, and each module is assembled in a certain order by molecular cloning technology to complete the L-cysteine ​​biosensor. Construction of biosensor expression plasmid pCcdRAe

[0032] The PCR method was used to amplify the structural gene of ccdR and its upstream and downstream nucleotide fragments using the Pantoea ananatis AJ13355 gene as a template. This process used primers ccdR for and ccdR re. Also use primers ccdA-efor and ccdA-e re to amplify the ccdA promoter fragment, and use primers ccd-efp for and ccd-efp re to amplify the eGFP gene fragment. Then, using primers ccdA-e for and ccd-efp re, the promoter nucleotide fragment of ccdA and the eGFP gene fragment were fused by fusion PCR. ccdR and its upstream and downstream nucle...

Embodiment 2

[0040] Example 2 Detection of L-cysteine ​​by L-cysteine ​​single-cell biosensor

[0041] In this example, the sensitivity and specificity of the biosensor to L-cysteine ​​were tested.

[0042] (1) A single colony of DH5α carrying the single-cell biosensor plasmid pCcdRAe was inoculated into a medium of LB+25 μg / L kanamycin, and cultured overnight at 37° C. at 200 rpm for 12 hours.

[0043] (2) Inoculate the overnight cultured bacterial solution into fresh LB+25 μg / L kanamycin medium at a ratio of 1:50, and culture overnight at 37° C. at 200 rpm for 12 hours.

[0044] (3) When the cell OD 600 When it reaches 0.5-0.8, add L-cysteine ​​with a final concentration of 0-32mmoL / L, and measure the fluorescence intensity after 2 hours.

[0045] (4) When measuring the fluorescence intensity, first centrifuge the bacteria, remove the supernatant, then wash the bacteria with PBS buffer and resuspend to make the resuspension OD 600 is about 0.5. A Hitachi fluorescence spectrophotometer ...

Embodiment 3

[0048] Example 3 Application of L-cysteine ​​single-cell biosensor in screening strains with high L-cysteine ​​production

[0049] In this example, the L-cysteine ​​single-cell biosensor is used in combination with a high-throughput screening system to screen for high-yield L-cysteine ​​strains ( Figure 5 ).

[0050] (1) Escherichia coli DH5α containing plasmid pCcdRAe was used as a starting strain for mutagenesis. The strain to be mutated was cultivated overnight at 37° C. and 200 rpm in a liquid medium of LB+25 μg / mL kanamycin. After washing with normal saline, the cells were resuspended and the OD 600 Adjust to 1.0, take 10 μL of bacterial solution for mutagenesis. For mutagenesis, ARTP was used to irradiate for 12-20s, and the cells after mutagenesis were cultured overnight in LB+25μg / L kanamycin liquid medium at 37°C and 200rpm;

[0051] (2) The cells cultured overnight were initially screened with a Beckman MoFlo XDP flow cytometer, and the cells with the fluorescen...

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Abstract

The invention discloses a preparation method of a biosensor for detecting L-cysteine and an application of the biosensor in detecting yield of escherichia coli L-cysteine. Main components of the biosensor comprise a transcriptional control gene CcdR coding gene from pantoea ananatis, a promoter region and a terminator region of the transcriptional control gene CcdR coding gene, a promoter region of a gene ccdA from pantoea ananatis, a green fluorescent protein coding gene eGFP and a plasmid framework pTrc-Mob. The biosensor can have a good linear relation under the L-cysteine concentration of 0-32 mmol L<-1> and has the highest fluorescence intensity value and cysteine concentration relation under the L-cysteine concentration of 0.01-8 mmol L<-1>. Through combination with a high-throughput screening system, the biosensor performs real-time detection on the yield of escherichia coli L-cysteine, can screen out high-yield mutants of L-cysteine and mutants for key enzymes in biological synthesis of L-cysteine. Specific, quantitative and real-time detection of L-cysteine can be realized, and the sensitivity is high.

Description

technical field [0001] The invention belongs to the technical field of biosensors, and relates to the construction and application of an L-cysteine ​​biosensor. Background technique [0002] L-cysteine ​​is an important sulfur-containing amino acid, which is widely used in food, medicine and cosmetic industries. Because it contains a highly reducing sulfhydryl group, L-cysteine ​​can be used as a natural reducing agent to break disulfide bonds in proteins. For example, the expectorant liquid with its derivative acetyl L-cysteine ​​as the main component can break the disulfide bond in the sputum to reduce the viscosity of sputum; another example, the perm liquid with L-cysteine ​​as the main component can Break the disulfide bond in hair, so it is widely used in the hairdressing industry; another example, L-cysteine ​​and its derivative glutathione can break the disulfide bond in tyrosinase to inactivate it, It can reduce the melanin produced by tyrosinase, so it is used in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12Q1/02C12N15/01G01N21/64C12R1/19
CPCC07K14/195C07K14/43595C12N15/01C12N15/70C12Q1/02G01N21/6486
Inventor 刘君刘川徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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