CN109679845B - Microbial cell factory constructed based on light-induced dielectrophoresis technology and application thereof - Google Patents

Abstract

The invention discloses a microbial cell factory constructed based on light-induced dielectrophoresis technology and its application, and belongs to the technical field of fermentation. The microbial cell factory constructed based on the light-induced dielectrophoresis technology of the present invention includes: an ITO sandwich mechanism, a cell factory mechanism arranged inside the ITO sandwich mechanism, an observation mechanism and a light operation mechanism; the cell factory mechanism includes a cell warehouse, a virtual conveyor belt, a cell culture mechanism Plate and cell plate racks. The invention can construct a cell factory at the micron scale, and simultaneously realize accurate and rapid screening of excellent strains and extraction of optimal parameters of the fermentation process in one factory. Compared with the traditional fermentation process, this method greatly shortens the cycle of strain screening and optimization of fermentation parameters, and the fine strains obtained from the cell level are more uniform and accurate, and the optimized parameters of fermentation can be closer to the essential characteristics of cells. And it is optimal, so as to improve fermentation efficiency and promote the development of microbial industry.

Description

Microbial cell factory based on light-induced dielectrophoresis technology and its application

technical field

The invention relates to a microbial cell factory constructed based on light-induced dielectrophoresis technology and its application, and belongs to the technical field of fermentation.

Background technique

Biofermentation engineering is an important part of biotechnology and an important link in the industrialization of biotechnology. It plays an increasingly important role in modern food, medicine and other high value-added industries. From a microscopic perspective, the essence of microbial fermentation engineering is the cycle of physiological metabolism and production and reproduction of fermenting bacteria in the colony starting from spore inoculation and shedding of the next spore. The product of its metabolism or reproduction process is the output of the fermentation industry. The whole fermentation process can be roughly divided into two parts: strain selection and colony fermentation.

The breeding of strains is to screen out the fermentation strains with the highest efficiency to the target product by microbiological methods. The current methods of strain screening can be mainly divided into two categories: i) improving new strains by genetic engineering; ii) extracting existing strains from nature. The former method uses genetic engineering technology to edit or recombine the genes that dominate part of the metabolic network of microbial cells, thereby inhibiting some non-important metabolic links or promoting the metabolic network closely related to the desired fermentation products, so that microbial cells tend to be more Metabolism, growth and reproduction are carried out in the direction that is beneficial to the fermentation output, so as to increase the fermentation output and improve the fermentation efficiency. The screening method of this type of strain can effectively screen out excellent strains with high output and develop some new types of strains. However, in the screening process, it not only requires the help of large-scale genetic engineering technology and corresponding equipment, and the cost It is not suitable for most small and medium-sized enterprises; the latter method is extracted from natural soil and other places, and the initially extracted soil contains a large amount of impurities and other strains, it is necessary to continuously cultivate the extract for a long time without interruption by means of bioimmunological methods. Although this kind of method has relatively low cost, its cycle is too long and the relative process is relatively complicated. In addition, this screening method has a certain degree of randomness, and the excellent strains screened are directly related to the soil.

Colony fermentation is the process of culturing the screened strains in a fermenter, controlling the input of raw materials, adjusting various external parameters in the process, and outputting the target product. Fermentation benefits are closely related to input raw materials and external parameters. In the traditional fermentation industry, relatively good fermentation parameters are obtained by detecting and comparing the benefits of fermentation products under the condition of constantly adjusting the input parameters of the input raw materials and various external factors. The fermentation parameters obtained by this method are actually not optimal fermentation parameters. On the other hand, because there are many parameters affecting the fermentation process, and the confidence interval of each influencing factor is large, it takes a long time and high cost to screen out suitable fermentation parameters by this fermentation experiment method, and it is impossible to screen out the most suitable fermentation parameters. optimal fermentation parameters. In addition, the traditional fermentation parameter optimization method is based on the clustering effect of the macro-fermentation process, and often ignores the individual differences between colony groups. fermentation process.

In addition, in today's fermentation industry, the selection of strains and the fermentation of colonies are often disconnected in practice, although the two processes complement each other. For example, the characteristics of strains in the process of strain selection have important guiding significance for the optimization of fermentation parameters in the process of colony fermentation, and the optimization of fermentation parameters can also promote the selection of optimal fermentation parameters, and these two steps are often independent. The two types of personnel are completed separately, lack of in-depth communication and mutual guidance.

SUMMARY OF THE INVENTION

The present invention aims at the shortcomings of long cycle, high cost, complicated procedures, etc. in the current microbial industry fermentation process, difficulty in obtaining optimal parameters in the process of colony fermentation, long parameter selection time, etc. The two processes are disconnected and other problems, and a method and equipment for constructing a microbial cell factory and realizing precise fermentation based on light-induced dielectrophoresis technology are provided.

The first object of the present invention is to provide a microbial cell factory constructed based on light-induced dielectrophoresis technology, including:

The ITO sandwich mechanism includes a first ITO glass and a second ITO glass; the first ITO glass includes a first glass substrate and a first ITO thin film layer disposed on the surface of the first glass substrate; the second ITO glass includes a second glass substrate, a second ITO thin film layer disposed on the second glass substrate, and a hydrogenated amorphous silicon layer disposed on the second ITO thin film layer; formed between the hydrogenated amorphous silicon layer and the first ITO thin film layer an intermediate layer; the intermediate layer includes an inlet and an outlet; a variable frequency alternating electric field is applied between the first ITO glass and the second ITO glass;

A cell factory mechanism, the cell factory mechanism is arranged in the middle layer, and the cell factory mechanism includes a cell warehouse, a virtual conveyor belt, a cell culture plate and a cell plate rack, and the cell plate rack passes through the virtual conveyor belt in the middle layer. Move between inlets, cell depots and cell culture plates;

an observation mechanism for observing or recording the operation process in the cell factory mechanism;

The optical operating mechanism is used for forming an optical module on the hydrogenated amorphous silicon layer to generate a non-uniform electric field.

Further, the intermediate layer includes a fixing glue for bonding the hydrogenated amorphous silicon layer and the first ITO thin film layer; the fixing glue forms a channel, and the channel is communicated with the inlet and the outlet of the middle layer , the inlet is used to input cells or cell plate racks, and the outlet is used to remove cells or cell culture plates.

Further, the cell warehouse contains several cells, and the cells are used for placing cell plate racks. When the cell plate rack is placed in the cell warehouse, the width of the cell plate rack decreases sequentially from top to bottom.

Further, the virtual conveyor belt is composed of several optical modules, and the virtual conveyor belt includes a first virtual conveyor belt for transferring the cell plate rack between the entrance of the intermediate layer and the cell warehouse, and a first virtual conveyor belt for transferring the cell plate rack between the cell warehouse and the cell culture plate. A second virtual conveyor belt for transferring cell plate racks between and a third virtual conveyor belt for placing the cell plate racks in or out of the cell warehouse. The first virtual conveyor belt and the second virtual conveyor belt move on a horizontal plane, and the third virtual conveyor belt moves on a vertical plane.

Further, the cell culture plate is formed by arranging several small squares of equal size.

Further, the observation mechanism is a CCD located above the ITO sandwich mechanism.

Further, the light operating mechanism includes a computer, a projector and an objective lens. A light module of a specific shape is generated on the computer, and the projector is used to map the light module to the entrance of the objective lens, and the objective lens gathers the light module into the ITO sandwich mechanism.

Further, the cell warehouse is manufactured by using 3D printing technology to print and solidify polyethylene glycol diacrylate hydrogel material layer by layer on the surface of hydrogenated amorphous silicon from bottom to top.

Further, the cell plate rack is made of 3D printing technology and polyethylene glycol diacrylate hydrogel material.

The second object of the present invention is to provide the application of the microbial cell factory constructed based on the light-induced dielectrophoresis technology in the selection of strains and the optimization of fermentation parameters.

The beneficial effects of the present invention are:

The invention can construct a cell factory at the micron scale, and simultaneously realize accurate and rapid screening of excellent strains and extraction of optimal parameters of the fermentation process in one factory. Compared with the traditional fermentation process, this method greatly shortens the cycle of strain screening and optimization of fermentation parameters, and the excellent strains obtained from the cell level are more uniform and accurate, and the optimized parameters of fermentation can be closer to the essential characteristics of cells. And it is optimal, so as to improve fermentation efficiency and promote the development of microbial industry.

Description of drawings

Figure 1 is a schematic diagram of the equipment for building a cell factory and precise fermentation based on light-induced dielectrophoresis technology;

Figure 2 is a schematic diagram of the ITO sandwich mechanism;

Figure 3 is a schematic diagram of the cell warehouse;

Figure 4 is a cell plate rack and it is placed on the cell depot;

Fig. 5 is a schematic diagram of a virtual traditional belt; (A) the optical module set at the control end of the PC; (B) a schematic diagram of the virtual conveyor belt in the ITO sandwich mechanism;

Figure 6 is a schematic diagram of the overall structure of the cell factory.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Example 1:

The principle of the equipment for constructing a microbial cell factory based on light-induced dielectrophoresis technology is shown in Figure 1. The device mainly includes four parts: observation part, ITO (indium tin oxide) sandwich mechanism part, microbial cell factory part and light operation part.

The observation part is mainly composed of a high-definition CCD and related software, which is used to observe or record the establishment of cell factories, microbiological cell screening, and cell plate racking operations. The ITO sandwich mechanism mainly includes two layers of ITO glass and an intermediate layer. The upper surface of the lower ITO glass is plated with a layer of hydrogenated amorphous silicon with a thickness of about 0.5-1 μm. Spaces such as cell factories and cell operations are isolated. ITO glass is coated with a layer of ITO film with a thickness of about 180nm on the glass surface. The ITO film has good electrical conductivity. In this structure, the upper and lower layers of ITO glass are used as two layers of conductive layers, and the ITO surfaces of the two layers of ITO glass are attached to on the middle layer. Hydrogenated amorphous silicon is a kind of photoelectric material. The entire ITO sandwich mechanism can form an electric field in the illumination area under the condition of an external AC electric field, and the force generated by the electric field is the photoinduced dielectrophoresis force. The light-induced dielectrophoretic force acting on the object in the middle layer can realize the screening and operation of the object. The microbial cell factory mainly includes: cell warehouse, virtual conveyor belt, cell culture plate and cell plate rack. The cell depot contains multiple layers, each layer has multiple cells of equal size, and each cell can hold a cell plate rack. The cell plate rack is made of colloidal material compatible with microbial cells by 3D printing technology, and is mainly used for loading microbial cells. Cell plate racks are available in a variety of sizes, each with equal lengths and thicknesses, but different widths. On the other hand, the cell racks placed on the cell depot gradually increase in width from bottom to top, so that the racks on each layer can be operated by light-induced dielectrophoresis force. The virtual conveyor belt is completely composed of two vertical boxes that are connected one by one and formed by the optical modules mapped in the middle layer. One is used to transfer cell racks between the cell depot and the cell culture plate, and the other is used to transfer the loaded cell racks to the cell depot. In addition, the two virtual conveyor belts are respectively moving at a constant speed along the conveying direction. A cell culture plate consists of a series of small squares of equal size, each of which can hold a cell plate rack. The cell plate rack can be directly removed from the gap of the fixed glue at the front end of the ITO sandwich mechanism for further expansion of culture, or it can also be directly added to the cell plate rack with cell culture raw materials and appropriate changes in fermentation conditions for direct fermentation to screen out cell fermentation. optimization parameters. The optical operation part mainly includes: computer control terminal, projector and objective lens, etc. In addition to controlling some precision moving mechanisms not listed in the system, the computer control terminal is mainly used to generate optical modules, such as: virtual conveyor belt; the projector maps the optical module to the entrance of the objective lens; the objective lens maps the optical module output by the projector Convergence in the ITO sandwich establishment.

In the process of realizing the construction and precise fermentation of the cell factory with this device, 1) the ITO sandwich mechanism was prepared with fixative glue as shown in Figure 1; 2) the cell warehouse was fabricated on the lower surface of the ITO sandwich mechanism by the micro-nano 3D printing technology 3) Place the ITO sandwich mechanism on the optical operating mechanism, adjust the position of the optical module and the ITO sandwich mechanism, so that the designed optical module can be mapped to the ITO sandwich mechanism 4) Inject an appropriate amount of microbial cells into the cell plate rack of the right end of the ITO sandwich mechanism; 5) Operate and screen the microbial cells by light-induced dielectrophoresis, and move the selected cells On the plate rack; 6) Move the cell plate rack loaded with microbial cells to the virtual conveyor belt, and use the conveyor belt to transfer it to the cell warehouse for storage or directly to the cell culture plate again; 7) Directly add microbes to the cell culture plate. Fermentation raw materials and changing fermentation parameters are optimized for fermentation parameters, and the cell culture plate can be removed from the gap of the fixed glue at the front end of the ITO sandwich mechanism.

The ITO sandwich mechanism is the basis for the implementation of the invention, and its structure is shown in Figure 2. The manufacturing process briefly includes the following steps: 1) On a 25mm×25mm×1mm transparent glass substrate, deposit a The ITO layer with a layer thickness of about 180nm constitutes the ITO glass; 2) Using the plasma enhanced chemical vapor deposition method, a layer of 0.5-1 μm thick hydrogenated amorphous silicon photoconductive layer material is plated on the surface of the ITO; 3) Using 3D microstructure The titration device drops the fixing glue on the surface of the hydrogenated amorphous silicon according to the designed structure, with a thickness of 100 microns, and pastes another layer of ITO glass on the upper surface of the fixing glue. As another preferred solution, it can also be directly cut with double-sided tape according to the set structure and size, and pasted on the surface of hydrogenated amorphous silicon, and then another layer of ITO glass is pasted on the other side of the double-sided tape to make ITO Sandwich Agency.

The cell warehouse is used to store the microbial cells that have been screened and placed on the cell plate. The schematic diagram of the cell warehouse structure is shown in Figure 3. In this embodiment, there are 3 layers in total, each layer has 3 grids, each grid can place a 500 μm×500 μm cell plate rack, and the layer height is about 50 μm. At the bottom of each grid is a bar with a width of about 30 μm and a thickness of about 2 μm, which is used to place the cell plate rack. As a priority plan, the cell warehouse is fabricated by 3D printing technology by printing and curing polyethylene glycol diacrylate hydrogel material layer by layer on the surface of hydrogenated amorphous silicon from bottom to top.

The cell plate rack is used to load microbial cells. As a preferred solution, the cell plate rack is also made of 3D printing technology and polyethylene glycol diacrylate hydrogel material. The cell plate rack has various specifications with a length of 500 μm, a height of 7-10 μm, a maximum width of 500 μm, and a gradual decrease of 50 μm. An example is shown in FIG. 4 . There are three sizes of cell plate racks in this example, 500 μm×500 μm, 500 μm×450 μm and 500 μm×400 μm. Cell plate racks with a size of 500 μm×500 μm are placed on the uppermost layer, those with a size of 500 μm×450 μm are placed in the middle layer, and those with a size of 500 μm×400 μm are placed in the lowest layer. And when all the cell plate racks are placed on the cell warehouse, the cell plate racks should be attached to the innermost part of the cell warehouse to ensure that the light from the bottom to the top can illuminate the cell plate racks of each layer, that is, the light-induced dielectric The swimming force can act on the cell plate rack of each layer.

A virtual conveyor belt is used to transport the cell plate rack from one end to the other, which is completely mapped into the ITO sandwich mechanism by the light module set at the PC end through projectors and objective lenses, etc. Because it is not an actual physical structure, but an optical image projection, it is called a "virtual conveyor belt" in this invention. In one embodiment, the optical module is arranged on the PC side according to FIG. 5(A). The optical module is mainly composed of two vertical square grids side by side, and the two vertical optical modules move alternately along its length direction; an additional static grid is set at the intersection of the two optical modules, using It is used to move the cell plate rack in the cell depot to the two vertical light modules. The width L of the optical module square is determined by the reduction ratio of the optical module at the control end of the PC and the pattern mapped to the ITO sandwich mechanism multiplied by the actual required size of the virtual conveyor belt. Fig. 5(B) is a virtual conveyor belt generated according to the light pattern of Fig. 5(A). The two optical modules are respectively mapped in the middle of the two vertical slits of the ITO sandwich mechanism. The horizontal conveyor belt is used to transfer the cell plate racks to the cell warehouse, and the vertical conveyor belt is used to transfer the cell plate racks in the cell warehouse to the cells. culture plate. The inner square width of each small square of the conveyor belt is slightly larger than 500 μm.

The overall structure of the cell factory is shown in Figure 6. In this embodiment, the two vertical slits of the ITO sandwich structure constitute all the space of the cell factory. In this space, the cell depot is fixed behind the front and rear slits on the surface of the hydrogenated amorphous silicon, and the cell factory has an inlet on the right side and an outlet on the front side. The inlet is mainly used for inputting cells and cell racks, and there is a gap near the inlet for screening microbial cells. With the help of light-induced dielectrophoresis, the microbial cells with good performance are screened out according to the mechanical or physiological characteristics of the microbial cells. , and move to the upper surface of the cell plate rack. A cell culture plate is placed at the exit position of the front side of the cell warehouse, and the cell culture plate can be used for directly culturing microbial cells, and the microbial cells can be taken out.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims (7)

1. a microbial cell factory based on light-induced dielectrophoresis technology, is characterized in that, comprises: The ITO sandwich mechanism includes a first ITO glass and a second ITO glass; the first ITO glass includes a first glass substrate and a first ITO thin film layer disposed on the surface of the first glass substrate; the second ITO glass includes a second glass substrate, a second ITO thin film layer disposed on the second glass substrate, and a hydrogenated amorphous silicon layer disposed on the second ITO thin film layer; formed between the hydrogenated amorphous silicon layer and the first ITO thin film layer an intermediate layer; the intermediate layer includes an inlet and an outlet; a variable frequency alternating electric field is applied between the first ITO glass and the second ITO glass; A cell factory mechanism, the cell factory mechanism is arranged in the middle layer, and the cell factory mechanism includes a cell warehouse, a virtual conveyor belt, a cell culture plate and a cell plate rack, and the cell plate rack passes through the virtual conveyor belt in the middle layer. Move between inlets, cell depots and cell culture plates; an observation mechanism for observing or recording the operation process in the cell factory mechanism; an optical operating mechanism for forming an optical module on the hydrogenated amorphous silicon layer to generate a non-uniform electric field; The cell warehouse includes a plurality of cells, and the cells are used for placing the cell plate rack, and when the cell plate rack is placed in the cell warehouse, the width of the cell plate rack decreases sequentially from top to bottom; The virtual conveyor belt is composed of several optical modules, and the virtual conveyor belt includes a first virtual conveyor belt for transferring cell plate racks between the entrance of the intermediate layer and the cell warehouse, and for transferring cells between the cell warehouse and the cell culture plate. a second virtual conveyor belt of the plate racks and a third virtual conveyor belt for placing the cell plate racks in or out of the cell warehouse; the first virtual conveyor belt and the second virtual conveyor belt move on a horizontal plane, and the The third virtual conveyor moves on the vertical plane. 2. a kind of microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, is characterized in that, described intermediate layer comprises and is used for bonding described hydrogenated amorphous silicon layer and the first ITO thin film layer The fixative glue; the fixative glue forms a channel, the channel is connected with the inlet and outlet of the middle layer, the inlet is used for inputting cells or cell plate racks, and the outlet is used for taking out cells or cell culture plates. 3 . The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1 , wherein the cell culture plate is formed by arranging several small squares of equal size. 4 . 4 . The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1 , wherein the observation mechanism is a CCD located above the ITO sandwich mechanism. 5 . 5. a kind of microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, is characterized in that, described light operating mechanism comprises computer, projector and objective lens, produces the light module of specific shape on the computer , the light module is mapped to the entrance of the objective lens through the projector, and the objective lens gathers the light module into the ITO sandwich mechanism. 6. A kind of microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, is characterized in that, described cell warehouse is the hydrogel material of polyethylene glycol diacrylate using 3D printing technology It is manufactured by layer-by-layer printing on the surface of hydrogenated amorphous silicon from bottom to top. 7. A kind of microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, it is characterized in that, described cell plate frame is made of polyethylene glycol diacrylate hydrogel material using 3D printing technology production.

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