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High-flux screening method based on biosensor

A biosensor and high-throughput technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of easy introduction of errors in fluorescent signals, difficult identification and sorting, background interference of fluorescent signals, etc.

Active Publication Date: 2019-04-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although biosensors detect intracellular target small molecule compounds and flow cytometry detects intracellular fluorescent signals with relatively high sensitivity, background interference of fluorescent signals, fluorescent signals are easy to introduce errors, and cells with small differences are difficult to identify and sort at the single cell level However, these factors greatly limit the screening efficiency of positive mutants; in addition, metabolites with important application value, including amino acids and aromatic compounds, are all secreted small molecule compounds, which are characterized in that the small molecule compounds are secreted in the cell Less internal accumulation, most of the compounds will be secreted extracellularly

Method used

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  • High-flux screening method based on biosensor
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  • High-flux screening method based on biosensor

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1: Shake flask culture simulates microdroplet-embedded biosensor cell amplification signal difference experiment

[0049] Since the cells in the test tube or shake flask fermentation come from the same parental cell, which is consistent with the single cell embedding and propagation mechanism in the droplet, we use the shake flask to culture the single cell to simulate the micro droplet embedding biosensor cells To amplify the signal difference, the specific design is as follows: the strains WJ60P (weak) and M9 (strong) with differences in 3-dehydroshikimic acid (3-dehydroshikimic acid, DHS) synthesis ability were used as experimental strains, and the two strains were shaken under the same conditions. Fermentation culture, initial inoculation concentration: 0.1OD600, medium: NBS, temperature: 37°C, rotation speed: 220rpm, fermentation culture for 8 hours, sampling at fixed points, samples were washed twice with PBS and resuspended to 1OD600, and then serially dil...

Embodiment 2

[0050] Example 2: Shake Flask Culture Simulated Microdroplet Encapsulation Biosensor Cell Feedback Mechanism Experiment

[0051] In order to simulate the cell feedback mechanism of microdroplet embedding biosensors, we used strains WJ60P (weak) and M9 (strong) with differential 3-dehydroshikimic acid synthesis ability as experimental strains, and carried out shake flask fermentation culture on the two strains under the same conditions , detect and observe the growth and fluorescence signal intensity changes of the two strains, compare the response fluorescence signal values ​​of the biosensor in the two strains, and verify the effect of the extracellular feedback of the secreted small molecule compound on the sensing of the biosensor. In this experiment, the initial inoculation concentration: 0.1OD600, medium: NBS, temperature: 37°C, rotation speed: 220rpm, fermentation culture for 8h, 11h, 14h, sampling at fixed points, the samples were washed twice with PBS and resuspended to...

Embodiment 3

[0052] Example 3: Development and construction of a genotype biosensor that responds to the metabolic small molecule compound 3-dehydroshikimic acid

[0053] In order to construct a genotype biosensor that responds to the metabolic small molecule compound 3-dehydroshikimic acid, first, we took the transcriptome of the 3-dehydroshikimic acid-free strain E.coli ATCC 8739 as a reference, and rationally constructed the experimental group. The transcriptome samples of the 3-dehydroshikimic acid-producing engineering strain fermented for 12h and 20h were analyzed, combined with RT-qPCR verification, and the transcriptional regulator gene cusR positively correlated with the change of 3-dehydroshikimic acid concentration was excavated, and the gene cusR and its promoter are the core part, and the 15-amino acid short peptide with the coding sequence of 5'-ggtggtggtggttctggtggtggtggatccggtggcggtggttct-3' is used as the connecting sequence to connect the induction gene cusR with the promo...

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Abstract

The invention discloses a high-flux screening method based on a biosensor, and belongs to the technical field of high-flux screening. A way of embedding biosensor-containing cells by liquid drops is adopted to carry out high-flux screening on a metabolite micromolecule 3-dehydrogenation shikimic acid cell factory to realize the efficient enrichment of 3-dehydrogenation shikimic acid high-yield bacterial strains. Compared with a single cell level screening method for combining the biosensor with a flow cytometer, on the one hand, the high-flux screening method disclosed by the invention utilizes the augmentable enrichment characteristics of cells in the liquid drops to enlarge an inter-unicellular fluorescence signal difference for N times to a difference among cell groups; on the other hand, through the characteristics that the liquid drops are used for isolating single cells and secretory products thereof, the influence of other cells and secretory products is eliminated, reductive cells and extracellular metabolite secreted by the reductive cells are truly communicated and fed back; through the above improvement, the screening sensitivity of the profitable positive clone bacterial strains can be effectively improved, and the enrichment efficiency of the profitable positive clone bacterial strains is improved.

Description

technical field [0001] The invention belongs to the field of high-throughput screening, and relates to a method for realizing high-throughput screening of cell factories producing metabolite small molecule compounds by using liquid droplets to embed cells containing biosensors. Background technique [0002] In recent years, with the continuous development of metabolic engineering and synthetic biology, more and more efficient microbial cell factories have been constructed to efficiently synthesize high value-added metabolites and small molecular compounds, including amino acids, proteins, organic acids, aromatic Compounds, etc. Compared with traditional chemical synthesis, the construction of microbial cell factories to synthesize small molecular compounds of metabolites is not only green and environmentally friendly, but also plays an increasing role in reducing production costs and improving corporate competitiveness. Therefore, using microbial cell factories to synthesize ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N13/00C12P7/42G01N21/64C12R1/19
CPCG01N21/6486C12N1/20C12N13/00C12P7/42
Inventor 涂然李梁坡王钦宏马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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