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D-psicose biosensor and application thereof

A technology of biosensor and psicose, which is applied in the field of synthetic biology and biochemical industry, can solve the problems of high equipment requirements, inability to real-time, low sensitivity, etc., achieve high-throughput screening, save time and labor costs

Pending Publication Date: 2022-04-12
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the following defects: (1) time-consuming and labor-intensive, generally the analysis time of each sample is greater than 30 minutes; (2) the requirements for instruments and equipment are high, because there is no ultraviolet signal, so a parallax detector needs to be used; (3) ) is low in sensitivity, and the detection limit of using a parallax detector is 0.5g / L; (4) the D-psicose in the cell cannot be analyzed online. Cannot analyze intracellular D-psicose in real time

Method used

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  • D-psicose biosensor and application thereof
  • D-psicose biosensor and application thereof
  • D-psicose biosensor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0024] Example 1D-Construction of psicose biosensor

[0025] In this example, a DNA-encoded D-psicose biosensor will be constructed.

[0026] Both the transcription factor PsiR and the inducible promoter PsiA were derived from Agrobacterium tumefaciens. The coding gene PsiR of PsiR was optimized and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (the optimized PsiR nucleotide sequence is shown in the sequence table SEQ ID NO.5), and cloned into the BamHI / HindIII digested vector pUC57 Between the sites, pUC57-PsiR was obtained; the promoter PsiA was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (the nucleotide sequence of PsiA is shown in the sequence table SEQ ID NO.7), and cloned into the BglII of the vector pUC57 / XbaI restriction site, to obtain pUC57-PsiA.

[0027] The expression intensity of transcription factors is a key factor affecting the response range of biosensors. The present invention uses 3 promoters (J23100, J23109 and J23110) of diffe...

Embodiment 2

[0039] D-Psicose Biosensor Analysis

[0040] The above nine kinds of plasmids carrying biosensors were transformed into Escherichia coli BL21(DE3). After overnight activation, they were transferred to LB+Kan medium (Table 3) according to the inoculum size of 3% (v / v). After culturing at 37°C and 200 rpm for 3 hours, the expression of the reporter gene was induced by adding D-psicose at a final concentration of 100 g / L; the blank control was set without adding D-psicose under the same conditions. Under the conditions of 37° C. and 200 rpm, the culture was continued for 5 h. Cells were collected by centrifugation, washed with PBS of 50 mM PBS (pH=7), and resuspended in PBS. The fluorescent signal of the reporter gene EGFP and the biomass OD of the cells were measured by a multi-functional microplate reader (TECAN Infinite Plex). 600 . When measuring the fluorescence signal of EGFP, the excitation light and emission light were set to 488nm and 520nm, respectively. Set the in...

Embodiment 3

[0047] Dynamic range analysis of D-psicose by biosensor

[0048] In order to analyze the dynamic range of the biosensor for D-psicose, the present invention selects J23109-PsiR-T7PsiO-EGFP and J23109-PsiR-PsiA02-EGFP which have higher sensitivity to 1g / L D-psicose. The induction method is as in Example 2, adding D-psicose with a final concentration of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0 g / L respectively, and measuring the fluorescence signal (excitation light and emission light) Light is 488nm and 520nm respectively) and biomass OD 600 , and use the pair OD 600 Normalize the fluorescent signal (A 520 / OD 600 ). The results showed that both J23109-PsiR-T7PsiO-EGFP and J23109-PsiR-PsiA02-EGFP responded to D-psicose within a certain range, and showed a substrate saturation effect after exceeding a certain range (results such as Figure 5 ). The dynamic range of J23109-PsiR-T7PsiO-EGFP is 0–4.0g / L D-psicose, and the dynamic range of J23109-PsiR-PsiA02-EGFP is 0–3....

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Abstract

The invention provides a D-psicose biosensor which can be used for detecting the content of D-psicose in vivo and in vitro of microorganisms, and belongs to the field of synthetic biology and biochemical engineering. The biosensor takes escherichia coli as a chassis, and consists of a constitutive promoter, a transcription factor PsiR, an inducible promoter and a reporter gene egfp. Wherein the constitutive promoter is used for controlling expression of a transcription factor PsiR, and the inducible promoter is used for controlling expression of a reporter gene egfp. When the D-psicose does not exist, the PsiR is combined with the regulation site PsiO of the inducible promoter to repress the transcription of a downstream reporter gene; when D-psicose exists, PsiR is not combined with PsiO any more, transcription repression of a downstream reporter gene is relieved, and a fluorescence signal is generated. In a certain range, the signal intensity of the reporter gene is in direct proportion to the logarithm of the concentration of the D-psicose, so that the concentration of the D-psicose can be calculated by monitoring the fluorescence signal intensity. According to the present invention, the binding site PsiO of the transcription factor FdeR is fused with the strong promoter to construct the artificial chimeric promoter, such that the sensitivity and the monitoring range of the sensor are optimized; the biosensor constructed by the invention not only can be used for monitoring the concentration of D-psicose in the environment, but also can be used for monitoring the yield of a D-psicose production strain, and has great application potential in the fields of biochemical engineering, industrial microorganisms and food.

Description

technical field [0001] The invention belongs to the fields of synthetic biology and biochemical industry, and in particular relates to a D-psicose biosensor and its application in food, fermentation and biochemical industry. Background technique [0002] As a rare sugar, D-psicose is difficult to be absorbed by the human gastrointestinal tract. It shows hypoglycemic and hypolipidemic properties beneficial to weight loss, and has important antioxidant activity. In addition, the U.S. Food and Drug Administration has listed D-psicose as generally recognized as safe (GRAS announcement, number GRN 498) in June 2014, allowing it to be used as a sweetener in the food industry and beverage manufacturing. [0003] Although D-psicose exists in nature, its content is extremely low, making separation and extraction very difficult and costly. At present, D-psicose is mainly synthesized by enzymatic conversion and microbial fermentation. However, since D-psicose itself is a colorless co...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/65C12N15/31G01N21/64C12R1/19
Inventor 吕永坤许敬亮朱丽娟张辉熊文龙阿拉牧屈凌波应汉杰王诗元
Owner ZHENGZHOU UNIV
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