WO2020147371A1

WO2020147371A1 - Microbial cell factory constructed on the basis of light-induced dielectrophoresis and applications thereof

Info

Publication number: WO2020147371A1
Application number: PCT/CN2019/114366
Authority: WO
Inventors: 李恭新; 刘飞; 陈珺
Original assignee: 江南大学
Priority date: 2019-01-16
Filing date: 2019-10-30
Publication date: 2020-07-23
Also published as: CN109679845A; CN109679845B

Abstract

The present invention relates to the technical field of fermentation. Disclosed are a microbial cell factory constructed on the basis of light-inducted dielectrophoresis and applications thereof. The microbial cell factory of the present invention constructed on the basis of light-inducted dielectrophoresis comprises: an ITO sandwich mechanism and, provided within the ITO sandwich mechanism, a cell factory mechanism, an observation mechanism, and a light operating mechanism. The cell factory mechanism comprises a cell warehouse, a virtual conveyor belt, a cell culturing plate, and a cell plate frame. The present invention allows the construction of a cell factory in the micrometer-scale level and simultaneously implements the accurate and quick screening of an excellent strain and the extraction of an optimal parameter for a fermentation process via one factory. Compared with a conventional fermentation process, the fermentation process of the present invention greatly shortens the cycle for strain screening and fermentation parameter optimization, an excellent strain acquired from a cell level is of increased uniformity and accuracy, and an optimized parameter acquired for fermentation approaches the nature of cells and is optimal, thus increasing fermentation benefits and promoting the development of the microbial industry.

Description

Microbial cell factory constructed 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

Biological fermentation engineering is an important part of current biotechnology and an important link in the industrialization of biotechnology. It is playing 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 repeated physiological metabolism and production and reproduction process of the fermenting bacteria in the colony from the spore inoculation to the next spore shedding. The product of the metabolism or reproduction process is the output of the fermentation industry. The whole fermentation process can be roughly divided into two parts: the breeding of strains and the fermentation of colonies.

The selection of strains is to screen out the fermentation strains with the highest efficiency for the target product through microbiological methods. The current methods of strain screening can be divided into two categories: i) new strains are improved through genetic engineering; ii) existing strains are extracted from nature. The former method uses genetic engineering technology to edit or reorganize 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 required fermentation products, and prompting the microbial cells to become more It is conducive to metabolism, growth and reproduction in the direction of fermentation output, thereby increasing fermentation output and improving fermentation efficiency. The screening method of this type of strain can effectively screen out excellent strains with high efficiency and develop some new types of strains. However, the screening process not only requires the use of large-scale genetic engineering technology and corresponding equipment, cost High, long cycle, and very high technical requirements for screening personnel, therefore, 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 lot of impurities And other bacterial species, it is necessary to rely on the method of biological immunology to cultivate the extract continuously for a long time without interruption. Although this kind of method is relatively low in cost, its cycle is too long and the process is relatively complicated. In addition, this screening method has a certain degree of randomness, and the selected excellent strains are directly related to the soil.

The fermentation of the colony is the process of cultivating the selected strains in the fermentor, 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. The traditional fermentation industry obtains relatively good fermentation parameters by detecting and comparing the benefits of fermentation products under the condition of continuously adjusting the input parameters of input raw materials and various external factors. The fermentation parameters obtained by this method are not the optimal fermentation parameters. On the other hand, because there are many parameters that affect 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 through this fermentation experiment method, and it is impossible to screen out the most suitable fermentation parameters. Excellent fermentation parameters. In addition, this traditional fermentation parameter optimization method is based on the clustering effect of the macro-fermentation process, and often ignores the personality differences between the colony groups, and it is difficult to obtain the dynamic changes in the characteristics of the bacterial colony during the growth and metabolism process, and cannot achieve precision and efficiency. The fermentation process.

In addition, in the current fermentation industry, the two processes of strain breeding and colony fermentation are actually often disconnected, although the two processes are complementary to each other. For example, the characteristics of the strains in the process of strain breeding have important guiding significance for the optimization of fermentation parameters in the colony fermentation process, and the optimization of fermentation parameters can also promote the selection of the optimal fermentation parameters, and these two steps are often independent The two types of personnel were completed separately, lacking in-depth communication and mutual guidance.

Summary of the invention

The present invention addresses the shortcomings of long cycle, high cost, complicated working procedures in the fermentation strain breeding process in the current microbial industry, the disadvantages of difficulty in obtaining optimal parameters in the colony fermentation process, long parameter selection time, etc., as well as strain breeding and colony fermentation The two processes are disconnected and other issues, providing a method and equipment for constructing microbial cell factories and realizing precise fermentation based on light-induced dielectrophoresis technology.

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

The cell factory mechanism, the cell factory mechanism is arranged in the middle layer, the cell factory mechanism includes a cell warehouse, a virtual conveyor belt, a cell culture plate, and a cell plate rack. The cell plate rack is placed in the middle layer by the virtual conveyor belt. Move between entrance, cell warehouse and cell culture plate;

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

The optical operating mechanism is used to form 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 communicates with the inlet and the outlet of the intermediate layer The inlet is used to input cells or cell plate racks, and the outlet is used to take out cells or cell culture plates.

Further, the cell warehouse includes a plurality of cells, and the cells are used for placing a cell plate rack. 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 a number of optical modules, and the virtual conveyor belt includes a first virtual conveyor belt for conveying 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 the cell plate racks in between and a third virtual conveyor belt for placing the cell plate racks in the cell warehouse or taking them 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 light module is mapped to the entrance of the objective lens through the projector, and the objective lens converges the light module into the ITO sandwich mechanism.

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

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 strain breeding and fermentation parameter optimization.

The beneficial effects of the invention are:

The invention can construct a cell factory at the micrometer scale, and simultaneously realize the accurate and rapid screening of excellent bacterial species and extract the optimal parameters of the fermentation process through a 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 the best to improve fermentation efficiency and promote the development of microbial industry.

BRIEF DESCRIPTION

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

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

Figure 3 is a simplified diagram of the cell warehouse;

Figure 4 shows the cell plate rack and its placement on the cell warehouse;

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

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

detailed description

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

Example 1:

The equipment principle of constructing a microbial cell factory based on light-induced dielectrophoresis technology is shown in Figure 1. The equipment mainly includes 4 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 process of cell factory establishment, microbiological cell screening, and cell plate rack operation. 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 0.5-1μm. The intermediate layer is fixed and fixed by the upper and lower ITO glass by a fixing glue. Isolate cell factories, cell operations and other spaces. ITO glass is coated with a layer of ITO film about 180nm thick on the surface of the glass. The ITO film has good electrical conductivity. In this structure, the upper and lower layers of ITO glass serve as two 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 structure can form an electric field in the illuminated area when an AC electric field is applied. The force generated by the electric field is the light-induced dielectrophoretic force. The light-induced dielectrophoretic force acting on the objects in the middle layer can realize the screening and operation of the objects. The microbial cell factory mainly includes: cell warehouse, virtual conveyor belt, cell culture plate and cell plate rack, etc. The cell warehouse 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 materials compatible with microbial cells through 3D printing technology, and is mainly used for loading microbial cells. The cell plate rack has many specifications, and the length and thickness of the various plate racks are equal, but the width is different. The cell plate rack placed on the cell warehouse gradually increases in width from bottom to top, so that each layer of the plate rack can be operated with light-induced dielectrophoresis force. The virtual conveyor belt is completely composed of two vertical boxes connected one by one and formed by the optical modules mapped on the middle layer. One is used to transfer the cell plate rack between the cell warehouse and the cell culture plate, and the other is used to transfer the cell plate rack loaded with cells to the cell warehouse. In addition, the two virtual conveyor belts are moving at a constant speed along the conveying direction. The cell culture plate is arranged by a series of small squares of equal size, and each small square can hold a cell plate rack. The cell plate rack can be directly removed from the gap of the fixed glue at the front 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 materials and the fermentation conditions appropriately changed for direct fermentation to screen out cell fermentation Optimized parameters. The light operation part mainly includes: computer control terminal, projector and objective lens, etc. In addition to controlling some precision movement 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 outputs the optical module of the projector Converged in the ITO sandwich organization.

In the process of using this device to realize the construction and precise fermentation of the cell factory, 1) Prepare the ITO sandwich structure with fixing glue as shown in Figure 1; 2) Create the cell warehouse on the lower surface of the ITO sandwich structure by micro-nano 3D printing technology And cell culture plate, and prepare the corresponding cell plate rack; 3) Place the ITO sandwich mechanism on the light operation mechanism, adjust the position of the light module and ITO sandwich mechanism, so that the designed light module can be mapped to the ITO sandwich mechanism Middle; 4) A cell plate rack in which an appropriate amount of microbial cells is injected into the gap of the fixing glue at the right end of the ITO sandwich mechanism; 5) The microbial cells are manipulated and screened by light-induced dielectrophoresis, and the selected excellent cells are moved to the cells 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) Add microbes directly to the cell culture plate Fermentation raw materials and fermentation parameters are changed to optimize the 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. Its structure is shown in Figure 2. The manufacturing process briefly includes the following steps: 1) Deposit a magnetron sputtering method on a transparent glass substrate of 25mm×25mm×1mm The ITO layer with a layer thickness of about 180nm constitutes ITO glass; 2) Using plasma enhanced chemical vapor deposition, a layer of hydrogenated amorphous silicon photoconductive layer material with a thickness of 0.5-1 μm is plated on the ITO surface; 3) Using 3D micro 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 sticks another layer of ITO glass on the upper surface of the fixing glue. As another preferred solution, you can also directly use double-sided tape to cut to the set structure and size, and paste it on the surface of hydrogenated amorphous silicon, and then stick another layer of ITO glass 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 structure of the cell warehouse is shown in Figure 3. In this embodiment, there are a total of 3 layers, each with 3 grids, each grid can hold a 500 μm×500 μm cell plate rack, and the layer height is about 50 μm. At the bottom of each grid there is a bar with a width of about 30μm and a thickness of about 2μm for placing the cell plate rack. As a preferred solution, the cell warehouse is made by 3D printing technology by printing and solidifying polyethylene glycol diacrylate hydrogel material 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 a variety of 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. In this embodiment, there are three cell plate racks with specifications of 500 μm×500 μm, 500 μm×450 μm, and 500 μm×400 μm. The cell plate rack with a size of 500μm×500μm is placed on the top layer, the cell plate rack with a size of 500μm×450μm is placed on the middle layer, and the cell plate rack with a size of 500μm×400μm is placed on the lowest layer. And when all 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 up can irradiate the cell plate racks on each layer, that is, the light-induced dielectric Swimming force can act on each layer of cell slab.

The virtual conveyor belt is used to transfer the cell plate rack from one end to the other end, and it is completely mapped into the ITO sandwich mechanism by the optical module set on the PC end through the projector and objective lens. Because it is not an actual physical structure, but only an optical image projection, it is called a "virtual conveyor belt" in this invention. In one embodiment, the optical module is set up on the PC side according to Figure 5(A). The optical module is mainly composed of two vertical square grids side by side. The two vertical optical modules move alternately along their length; an additional static grid is set at the intersection of the two optical modules. To move the cell plate rack in the cell warehouse to two vertical light modules. The width L of the optical module grid is determined by the reduction ratio of the optical module at the PC control end and the pattern mapped to the ITO sandwich structure multiplied by the actual size of the virtual conveyor belt. Fig. 5(B) is a virtual conveyor belt produced according to the light pattern of Fig. 5(A). The two light modules are respectively mapped in the two vertical gaps of the ITO sandwich mechanism. The horizontal conveyor belt is used to transport the cell slabs to the cell warehouse, and the vertical conveyor belt is used to transport the cell slabs in the cell warehouse to the cells. Culture plate. The width of the square in 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 mechanism constitute all the spaces of the cell factory. In this space, the cell warehouse is fixed behind the front and rear gaps on the surface of the hydrogenated amorphous silicon. There is an entrance on the right side of the cell factory and an exit on the front side. The import is mainly used for importing cells and cell slabs, and there is a gap near the entrance for screening microbial cells. With the help of light-induced dielectrophoresis, the microbial cells with good performance are screened out according to their mechanical or physiological characteristics. , And move to the upper surface of the cell plate rack. A cell culture plate is placed at the front exit of the cell warehouse. The cell culture plate can be used to directly cultivate microbial cells and can take out the microbial cells.

The above-mentioned embodiments are only preferred embodiments for fully explaining the present invention, and the protection scope of the present invention is not limited thereto. The equivalent substitutions or changes 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

A microbial cell factory constructed based on light-induced dielectrophoresis technology, characterized in that it 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 film layer disposed on the surface of the first glass substrate; the second ITO glass includes The second glass substrate, the second ITO thin film layer provided on the second glass substrate, and the hydrogenated amorphous silicon layer provided on the second ITO thin film layer; formed between the hydrogenated amorphous silicon layer and the first ITO thin film layer Intermediate layer; the intermediate layer includes an inlet and an outlet; a variable frequency AC electric field is applied between the first ITO glass and the second ITO glass;

The cell factory mechanism, the cell factory mechanism is arranged in the middle layer, the cell factory mechanism includes a cell warehouse, a virtual conveyor belt, a cell culture plate, and a cell plate rack. The cell plate rack is placed in the middle layer by the virtual conveyor belt. Move between entrance, cell warehouse and cell culture plate;

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

The optical operating mechanism is used to form an optical module on the hydrogenated amorphous silicon layer to generate a non-uniform electric field.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the intermediate layer includes a fixing for bonding the hydrogenated amorphous silicon layer and the first ITO film layer. Glue; the fixation glue forms a channel, the channel is connected with the inlet and the outlet of the intermediate layer, the inlet is used to input cells or cell plate rack, and the outlet is used to take out cells or cell culture plates.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the cell warehouse comprises a plurality of cells, and the cells are used to place a cell plate rack, and the cell plate When the rack is placed in the cell warehouse, the width of the cell plate rack decreases from top to bottom.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the virtual conveyor belt is composed of a plurality of light modules, and the virtual conveyor belt includes an entrance and a cell warehouse at the middle level. The first virtual conveyor belt that transfers the cell plate racks between, the second virtual conveyor belt that is used to transfer the cell plate racks between the cell warehouse and the cell culture plate, and the cell plate racks used to place or take the cell plate racks out of the cell warehouse The third virtual conveyor belt; 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.
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.
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.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the light operating mechanism includes a computer, a projector and an objective lens, and a light module with a specific shape is generated on the computer. The projector maps the light module to the entrance of the objective lens, and the objective lens converges the light module into the ITO sandwich structure.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the cell warehouse is a 3D printing technology to hydrogenate polyethylene glycol diacrylate hydrogel materials The surface of the amorphous silicon is printed layer by layer from bottom to top and solidified layer by layer.
The microbial cell factory constructed based on light-induced dielectrophoresis technology according to claim 1, wherein the cell plate rack is made of polyethylene glycol diacrylate hydrogel material using 3D printing technology .