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Flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof

A flow cytometry and high-throughput technology, which is applied in the field of high-throughput screening kits for ethanol-producing fungi based on flow cytometry, can solve problems such as large workload and reduce screening efficiency, so as to improve efficiency and save labor. The effect of low cost and reagent quantity

Inactive Publication Date: 2015-04-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have a large workload. When screening colonies, they often rely on the experience of experimenters and rely on naked eye observation to select the target strain. There is blindness, which greatly reduces the screening efficiency.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Determination of Effect of Ion Beam Implantation on ROS Content in Saccharomyces cerevisiae Using the Kit of the Invention

[0040] 1. Configuration solution

[0041] Reagent B: propidium iodide (PI): 0.3 mg; 2,7-dihydrodichlorofluorescein yellow sodium diacetate (DCFH-DA): 0.3 mg, packed in brown centrifuge tubes, kept away from light, added without Dilute the volume to 3mL with bacterial double-distilled water, filter and sterilize in the dark, and use it immediately;

[0042] 2. Determination of the effect of ion beam implantation on ROS content in Saccharomyces cerevisiae

[0043] 1. Ion Beam Implantation of Saccharomyces cerevisiae

[0044] (a) Centrifuge the cultured bacterial solution at 4°C, 5500r / min for 10min, discard the supernatant, wash with reagent A twice, then suspend the yeast cell sediment with reagent A, and set aside for N + Injection processing.

[0045] (b) Dilute the above bacterial suspension with a sterilized protective solution (0.5% (wt) s...

Embodiment 2

[0056] Using the kit of the present invention to measure the influence of ultraviolet irradiation mutagenesis on ROS content in Saccharomyces cerevisiae

[0057] 1. Configuration solution

[0058] Reagent B: propidium iodide (PI): 0.3 mg; 2,7-dihydrodichlorofluorescein yellow sodium diacetate (DCFH-DA): 0.3 mg, packed in brown centrifuge tubes, kept away from light, added without Dilute the volume to 3mL with bacterial double-distilled water, filter and sterilize in the dark, and use it immediately;

[0059] 2. Determination of the effect of ultraviolet radiation on the ROS content in Saccharomyces cerevisiae

[0060] 1. UV Irradiation of Saccharomyces cerevisiae

[0061] (a) Centrifuge the cultured bacterial solution at 4°C, 5500r / min for 10min, discard the supernatant, wash with reagent A twice, then suspend the yeast cell sediment with reagent A, and wait for UV irradiation injection.

[0062] (b) Dilute the bacterial suspension in step (a) with sterile water, take 2 mL ...

Embodiment 3

[0073] High-throughput screening of ethanol-producing Saccharomyces cerevisiae by using the kit of the present invention

[0074] 1. Mutagenesis of Saccharomyces cerevisiae

[0075] The starting strain Saccharomyces cerevisiae S23 (the starting strain used was screened in the laboratory, has been reported in the patent 201410495426.9, and the present invention is only used as an example, and does not involve the protection of specific strains) was made into a cell suspension, and the cell concentration was adjusted to 10 6 cells / ml, then centrifuge the bacterial solution at 4°C, 5500r / min for 10min, discard the supernatant, wash with reagent A twice, then suspend the yeast cell sediment with reagent A for mutagenesis treatment. The mutagenesis treatment is as follows: different ultraviolet irradiation intensity irradiation treatment, different ion beam implantation treatment, and then a total of 48 parts of different mutagenic bacteria solutions are selected.

[0076] 2. Kit ...

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Abstract

The invention discloses a flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof. The kit comprises a box body, reagents, a cell culture plate, a centrifuge tube and a copper screen. The reagents are as follows: 1000 mL of reagent A composed of 6.15g of citric acid and 17.544g of sodium citrate, reagent B composed of 0.3mg of propidium iodide (PI) and 0.3mg of DCFH-DA, 50 mL of reagent C composed of 2g of glucose, 50 mL of reagent D composed of 10g of glucose, 50 mL of reagent E (pH value is 5.5) composed of 0.5g of yeast powder, 0.5g of tryptone, 0.05g of ammonium monoacid phosphate and 0.025g of magnesium sulfate, and 50 mL of reagent F (pH value is 5.5) composed of 0.05g of yeast powder, 0.05g of tryptone, 0.05g of ammonium monoacid phosphate and 0.025g of magnesium sulfate.

Description

Technical field [0001] The invention is the field of microbial fermentation technology, which involves a kit and its application based on the high -throughput of flow -based cell technology high -throughput. Background technique [0002] Biomass energy is an important part of the future sustainable energy system. Since this century, biological can enter the research field of renewable resources. It has become the main direction of energy in the world with high pollution and high efficiency. ThereforeBiological energy has become the common goal of various countries.Among biological energy, the most widely studied, the most effective is the use and development of biological ethanol.From the perspective of the current level of science and technology and development of the society, fuel ethanol is fully possible to realize industrial production in the foreseeable next decades. [0003] Excellent microbial bacteria are the foundation and key to the fermentation industry. To make the t...

Claims

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Application Information

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IPC IPC(8): C12N1/14
CPCC12N1/14
Inventor 虞龙刘媛李玉燕
Owner NANJING UNIV OF TECH
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