Micro-fluidic chip photostimulation device, yeast single-cell photoregulation gene expression method and application

A microfluidic chip and yeast cell technology, applied in stress-stimulated microbial growth methods, chemical instruments and methods, and microbial-based methods, can solve problems such as slow effects, high requirements for experimental methods, and time resolution problems

Pending Publication Date: 2021-06-04
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among these important methods and technologies, gene perturbation method changes the phenotype of cells through gene knockout, gene overexpression and gene mutation. However, because this method acts on the whole cell and the effect is slow, it is more inclined to destroy intracellular molecular interactions Therefore, this method cannot be used to explore the molecular regulation mechanism inside the cell; the chemical substance perturbation method disturbs the concentration of intracellular substances by chemical substances, Induce or turn off gene expression, however, chemical perturbation methods cannot focus on a specific single cell, and also suffer from temporal resolution, because the reversal of chemical reaction perturbation requires physical changes in solution conditions; microinjection technology itself is very demanding on experimental methods , the experiment is difficult, and it is a low-throughput invasive technique, and it is only effective for specific cells

Method used

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  • Micro-fluidic chip photostimulation device, yeast single-cell photoregulation gene expression method and application
  • Micro-fluidic chip photostimulation device, yeast single-cell photoregulation gene expression method and application
  • Micro-fluidic chip photostimulation device, yeast single-cell photoregulation gene expression method and application

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Experimental program
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Effect test

Embodiment 1

[0072] A microfluidic chip light stimulation device, such as figure 1 As shown, the device includes a cell culture chip 1, a culture control unit and a light stimulation unit for maintaining the growth of living cells. The cell culture chip 1 includes a micro-table array 101 for capturing cells, a glass bottom plate 103 and an intermediate channel 102 for simulating blood vessel circulation. The CAD top view of the cell culture chip 1 is as follows: figure 2As shown, each cell culture chip 1 contains six independent channels. The micro-stage array 101 is made of polydimethylsiloxane, and the gap between it and the glass bottom plate 103 is 4 microns. The cell culture chip 1 is placed on a motorized stage 2 . The culture control unit includes a cage constant temperature incubator 3, which provides a constant temperature environment for the cultivation of yeast cells. The culture control unit also includes a first microinjection pump 5 and a second microinjection pump 6 comm...

Embodiment 2

[0074] The cell density of Saccharomyces cerevisiae W303-1a was about 5.5×10 after overnight culture by 600 nm absorbance measurement. 5 cells / ml, inject the yeast cell suspension into the microfluidic cell culture chip at a flow rate of 0.5 ml / min, and observe the cell capture situation through a microscope. Then the yeast extract powder peptone glucose medium was passed through at a flow rate of 0.1 ml / min, and at 30° C., the yeast growth image was imaged under bright-field LED light source as the lighting condition. The result is as image 3 shown. image 3 In the middle, the time in the upper left corner indicates the elapsed time from the introduction of the yeast culture medium, and the white cutting head indicates that the yeast cells with smaller cell diameters, such as newly divided progeny cells, are separated from the micro-stage array 101 of the cell culture chip 1, so no Can cause cell accumulation problems. from image 3 It can be seen that the yeast cells ca...

Embodiment 3

[0076] Using homologous recombination to construct light-stimulus-responsive Saccharomyces cerevisiae cell samples, the schematic diagram is as follows Figure 4 shown. Using PIF3 protein to link the VP16 activation domain constitutes the first module, and NPhyB and the lexA-DBD DNA binding domain constitute the second module. NPhyB undergoes a conformational change under the stimulation of 660nm laser and binds to PIF3 to activate downstream gene expression. However, under the stimulation of far-infrared light at 740 nanometers, NPhyB and PIF3 were dissociated, and the expression of downstream genes was stopped.

[0077] The specific process is as follows:

[0078] (1) Construction of transfection plasmids comprising PIF3-VP16, NPhyB-lexA-DBD and TATAbox-EGFP;

[0079] (2) Configure 1 mol / L lithium acetate to prepare yeast competent cells, and mix the transfection plasmid and competent cells at 42°C for one minute;

[0080] (3) Screen positive clones under appropriate scr...

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Abstract

The invention relates to a micro-fluidic chip photostimulation device, a yeast single-cell photoregulation gene expression method and application. The micro-fluidic chip photostimulation device comprises a cell culture chip, a culture control unit for maintaining the growth of living cells, and a photostimulation unit. According to the method for performing yeast single-cell photoregulation gene expression by adopting the micro-fluidic chip photostimulation device, the micro-fluidic chip device is adopted for performing microscope measurement on yeast cells, fixed-point quantitative photostimulation can be performed on the single cells at a single cell level, and yeast specific gene expression is started, and signal change caused by the change of the yeast gene expression quantity is collected for a long time. By adopting the method, statistical analysis can be carried out on cell heterogeneity response by wide stimulation on a plurality of yeast cells, a state space dynamic track corresponding to a cell response process is comprehensively provided, and average information loss of a population response result is avoided.

Description

technical field [0001] The invention belongs to the technical field of gene regulation, and in particular relates to a microfluidic chip light stimulation device and a yeast single-cell light-regulated gene expression method and application. Background technique [0002] In the study of gene circuits, due to the randomness of biological systems, the states of cells are randomly distributed in the state space. Observation at the single-cell level can give the state of each cell, and statistics on a large number of single-cell states can obtain the state distribution of cells in a certain period of time and in a certain environment. This state distribution strictly describes all the information of the cell in this situation. However, the population response of cells can only give the average value of the cell state, and cannot reflect the relationship between different states in the high-dimensional state space of cells, and may even fail to distinguish two overlapping states...

Claims

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Application Information

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IPC IPC(8): C12M1/42C12M1/38C12M1/36C12M1/34C12M1/26C12M1/00B01L3/00C12N15/81C12N15/65C12N13/00C12R1/865
CPCC12M23/16C12M23/20C12M29/00C12M33/04C12M35/02C12M41/14C12M41/46C12M41/08C12M41/48B01L3/5027C12N15/81C12N15/65C12N13/00C12N2830/002
Inventor 刘传波刘琼汪劲
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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