WO2020147371A1 - 基于光诱导介电泳技术构建的微生物细胞工厂及其应用 - Google Patents

基于光诱导介电泳技术构建的微生物细胞工厂及其应用 Download PDF

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WO2020147371A1
WO2020147371A1 PCT/CN2019/114366 CN2019114366W WO2020147371A1 WO 2020147371 A1 WO2020147371 A1 WO 2020147371A1 CN 2019114366 W CN2019114366 W CN 2019114366W WO 2020147371 A1 WO2020147371 A1 WO 2020147371A1
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cell
light
ito
conveyor belt
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李恭新
刘飞
陈珺
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass

Definitions

  • 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.
  • 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.
  • 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.
  • 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 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 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 two processes of strain breeding and colony fermentation are actually often disconnected, although the two processes are complementary to each other.
  • 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.
  • 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;
  • 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 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.
  • the cell warehouse includes a plurality of cells, and the cells are used for placing a cell plate rack.
  • the width of the cell plate rack decreases sequentially from top to bottom.
  • 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.
  • 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 cell culture plate is formed by arranging several small squares of equal size.
  • observation mechanism is a CCD located above the ITO sandwich mechanism.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • ITO Indium Tin Oxide
  • 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 0.5-1 ⁇ m.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • At the bottom of each grid there is a bar with a width of about 30 ⁇ m and a thickness of about 2 ⁇ m for placing the cell plate rack.
  • 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.
  • 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 ⁇ m, a height of 7-10 ⁇ m, a maximum width of 500 ⁇ m, and a gradual decrease of 50 ⁇ m. An example is shown in FIG. 4. In this embodiment, there are three cell plate racks with specifications of 500 ⁇ m ⁇ 500 ⁇ m, 500 ⁇ m ⁇ 450 ⁇ m, and 500 ⁇ m ⁇ 400 ⁇ m.
  • the cell plate rack with a size of 500 ⁇ m ⁇ 500 ⁇ m is placed on the top layer, the cell plate rack with a size of 500 ⁇ m ⁇ 450 ⁇ m is placed on the middle layer, and the cell plate rack with a size of 500 ⁇ m ⁇ 400 ⁇ m is placed on the lowest layer.
  • 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.
  • 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 ⁇ m.
  • the overall structure of the cell factory is shown in Figure 6.
  • the two vertical slits of the ITO sandwich mechanism constitute all the spaces of the cell factory.
  • the cell warehouse is fixed behind the front and rear gaps on the surface of the hydrogenated amorphous silicon.
  • the import is mainly used for importing cells and cell slabs, and there is a gap near the entrance for screening microbial cells.
  • the microbial cells with good performance are screened out according to their mechanical or physiological characteristics.
  • 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.

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Abstract

本发明公开了基于光诱导介电泳技术构建的微生物细胞工厂及其应用，属于发酵技术领域。本发明的基于光诱导介电泳技术构建的微生物细胞工厂，包括：ITO三明治机构、设置在ITO三明治机构内部的细胞工厂机构、观测机构和光操作机构；细胞工厂机构包括细胞仓库、虚拟传送带、细胞培养板和细胞板架。本发明能够在微米尺度层面构建细胞工厂，并在通过一个工厂里同时实现优良菌种的精确快速的筛选和提取出发酵过程的最优参数。相对于传统的发酵过程，本发明的发酵过程极大地缩短了菌种筛选和发酵参数优化的周期，并且从细胞层面获得的优良菌种更均一、更准确，获得发酵的优化参数更能接近于细胞本质特性并且是最优的，以此提高发酵效益，推动微生物工业的发展。

Description

基于光诱导介电泳技术构建的微生物细胞工厂及其应用 技术领域
本发明涉及一种基于光诱导介电泳技术构建的微生物细胞工厂及其应用，属于发酵技术领域。
背景技术
生物发酵工程是现在生物技术中的重要组成部分，是生物技术产业化的重要环节，其在现代食品、医药等高附加值工业中所发挥的作用越来越大。从微观层面看，微生物发酵工程的实质是菌落中的发酵菌从芽孢接种开始在下一个芽孢脱落的周而复始的生理代谢和生产繁殖过程，其代谢或繁殖过程中的产物即为发酵工业的产出。整个发酵过程可以粗略地分成两部分：菌种的选育和菌落的发酵。
菌种的选育是通过微生物学的方法筛选出对目标产物效率最高的发酵菌种。现今菌种筛选的方法主要可分为两类：i)通过基因工程的方法改良出新菌种；ii)从自然界中提取已有的菌种。前一种方法借助基因工程技术对主导微生物细胞的部分代谢网络的基因进行编辑或重组，从而抑制部分非重要的代谢环节或者促进与所需发酵产物密切相关的代谢网络，促使微生物细胞趋向于更有利于发酵产出的方向进行代谢、生长和繁殖，以此增加发酵产出提高发酵效率。该类菌种的筛选方法能够有效筛选出具有高效产出的优良菌种和开发出一些新类型的菌种，然而，其在筛选过程中不仅需要借助大型的基因工程技术相应的仪器设备，成本高、周期长，而且对筛选人员的技术要求也非常高，因此，不适用于大部分的中小型企业；后一种方法从自然界的土壤等地方提取，初始提取出的土壤中含有大量的杂质和其他菌种，需要借助于生物免疫学的方法通过对提取物连续多次不间断地长时间培育。这类方法虽然成本相对较低，但是其 周期太长、相对工序较复杂。此外，这种筛选方法有一定的随机性，所筛选的优良菌种与所取的土壤有直接关系。
菌落的发酵是将筛选出的菌种培养在发酵罐中，通过控制原料的输入、调节过程中的各种外界参数，并输出目标产物的过程。发酵效益与输入原料和外界参数密切相关。传统的发酵工业是在不断调控输入原料和各种外界因素的输入参数情况下，通过检测和比较发酵产物的效益来获得相对较好的发酵参数。这种方法获得的发酵参数其实并不是最优的发酵参数。另一方面，由于影响发酵过程的参数较多，并且每个影响因素的置信区间较大，通过这种发酵实验的方法来筛选出合适的发酵参数需要的时间长、成本高，无法筛选出最优的发酵参数。此外，传统这种发酵参数优化方法都是基于宏观发酵过程的聚群效应，而往往忽略了菌落群体间的个性差异，而难以获得针对菌群生长代谢过程中特性动态的变化，无法实现精准高效地发酵过程。
此外，在现今的发酵工业里，菌种的选育和菌落的发酵在实际上两个过程往往是脱节的，尽管两个过程相辅相成。例如：菌种选育过程中菌种的特性对菌落发酵过程中发酵参数的优化具有重要的指导意义，而发酵参数的优化也能促进筛选出最优发酵参数，而这两个步骤往往由独立的两类人员分别完成，缺乏深入的沟通和相互指导。
发明内容
本发明针对当前微生物工业中发酵菌种选育过程中周期长、成本高、工序复杂等不足，菌落发酵过程中难以获得最优参数、参数选择时间长等缺点，以及菌种选育与菌落发酵两个过程脱节等问题，提供一种基于光诱导介电泳技术构建微生物细胞工厂和实现精准发酵的方法和设备。
本发明的第一个目的是提供一种基于光诱导介电泳技术构建的微生物细胞工厂，包括：
ITO三明治机构，包括第一ITO玻璃和第二ITO玻璃；所述的第一ITO玻璃包括第一玻璃基底和设置在第一玻璃基底表面的第一ITO薄膜层；所述的第二ITO玻璃包括第二玻璃基底、设置在第二玻璃基底上的第二ITO薄膜层和设 置在第二ITO薄膜层上的氢化非晶硅层；所述氢化非晶硅层与第一ITO薄膜层之间形成中间层；所述中间层包括一个入口和一个出口；在第一ITO玻璃和第二ITO玻璃之间施加可变频率的交流电场；
细胞工厂机构，所述的细胞工厂机构设置在所述中间层内，所述细胞工厂机构包括细胞仓库、虚拟传送带、细胞培养板和细胞板架，所述细胞板架通过虚拟传送带在中间层的入口、细胞仓库和细胞培养板之间移动；
观测机构，用于观测或记录所述细胞工厂机构中的操作过程；
光操作机构，用于在所述氢化非晶硅层上形成光模块，产生非均匀电场。
进一步地，所述的中间层包括用于粘合所述氢化非晶硅层与第一ITO薄膜层的固定胶；所述固定胶形成一条通道，所述通道与中间层的入口和出口相连通，所述入口用于输入细胞或细胞板架，所述出口用于取出细胞或细胞培养板。
进一步地，所述的细胞仓库包含若干单元格，所述单元格用于放置细胞板架。所述细胞板架在细胞仓库中放置时，从上往下，细胞板架的宽度依次减小。
进一步地，所述的虚拟传送带由若干光模块构成，所述虚拟传送带包括用于在中间层入口与细胞仓库之间传送细胞板架的第一虚拟传送带、用于在细胞仓库与细胞培养板之间传送细胞板架的第二虚拟传送带和用于将细胞板架置于细胞仓库或从细胞仓库中取出的第三虚拟传送带。所述的第一虚拟传送带和第二虚拟传送带在水平面上移动，所述的第三虚拟传送带在铅直面上移动。
进一步地，所述的细胞培养板由尺寸相等的若干小方格排列形成。
进一步地，所述的观测机构是位于所述ITO三明治机构上方的CCD。
进一步地，所述的光操作机构包括电脑、投影仪和物镜，在电脑上产生特定形状的光模块，通过投影仪将光模块映射到物镜入口处，物镜将光模块汇聚到ITO三明治机构中。
进一步地，所述的细胞仓库是用3D打印技术将聚乙二醇二丙烯酸酯的水凝胶材料在氢化非晶硅表面从下往上逐层打印逐层固化制造而成的。
进一步地，所述的细胞板架是用3D打印技术和聚乙二醇二丙烯酸酯水凝胶材料制成。
本发明的第二个目的是提供所述的基于光诱导介电泳技术构建的微生物细胞工厂在菌种选育和发酵参数优化中的应用。
本发明的有益效果是：
本发明能够在微米尺度层面构建细胞工厂，并在通过一个工厂里同时实现优良菌种的精确快速的筛选和提取出发酵过程的最优参数。相对于传统的发酵过程，该方法极大地缩短了菌种筛选和发酵参数优化的周期，并且从细胞层面获得的优良菌种更均一、更准确，获得发酵的优化参数更能接近于细胞本质特性并且是最优的，以此提高发酵效益，推动微生物工业的发展。
附图说明
图1为基于光诱导介电泳技术构建细胞工厂和精准发酵的设备原理图；
图2为ITO三明治机构简图；
图3为细胞仓库简图；
图4为细胞板架及其被放置在细胞仓库上；
图5为虚拟传统带简图；(A)PC机控制端设置的光模块；(B)ITO三明治机构内的虚拟传送带简图；
图6为细胞工厂整体结构示意图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：
基于光诱导介电泳技术构建微生物细胞工厂的设备原理如图1所示。该设备主要包括4个部分：观测部分、ITO(氧化铟锡)三明治机构部分、微生物细胞工厂部分和光操作部分。
观测部分主要是由一台高清CCD及相关软件组成，用于观测或记录细胞工厂建立、微生物学细胞筛选、细胞板架操作等过程。ITO三明治机构主要包括两层ITO玻璃以及中间层，其中在下层的ITO玻璃上表面镀了一层厚为0.5-1μm左右的氢化非晶硅，中间层由固定胶将上下层的ITO玻璃固定并隔离出细胞工 厂、细胞操作等空间。ITO玻璃是在玻璃表面镀了一层180nm左右厚的ITO薄膜，ITO薄膜具有良好导电特性，在该结构中上下两层ITO玻璃作为两层导电层，并且两层ITO玻璃的ITO面都贴在中间层上。氢化非晶硅是一种光致电材料，整个ITO三明治机构在外加交流电场情况下在光照区域能够形成电场，该电场产生的力即为光诱导介电泳力。光诱导介电泳力作用于中间层的物体能够实现对物体的筛选和操作等。微生物细胞工厂主要包括：细胞仓库、虚拟传送带、细胞培养板和细胞板架等。细胞仓库包含多层，每层有尺寸相等的多个单元格，每个单元格能够放置一块细胞板架。细胞板架是用微生物细胞兼容的胶体材料通过3D打印技术制成，主要用于装载微生物细胞。细胞板架有多种规格，各种板架的长度和厚度相等，但宽度不一。而在放置在细胞仓库上的细胞板架，从下往上宽度逐渐增加，使得每一层的板架都能够用光诱导介电泳力进行操作。虚拟传送带完全由映射在中间层的光模块构成的一个个连接在一块的两条垂直的方框组成。一条用于在细胞仓库与细胞培养板之间传送细胞板架，另一条用于将装载好细胞的细胞板架传输到细胞仓库。此外，两条虚拟传送带分别沿着传输方向匀速移动着。细胞培养板由尺寸相等的一系列小方格排列而成，每个小方格力能够放置一块细胞板架。细胞板架能够从ITO三明治机构前端固定胶的空隙处直接移出用于进一步的扩大培养，或者也能够直接在细胞板架内加入细胞培养原料和适当改变发酵条件进行直接发酵，以筛选出细胞发酵的优化参数。光操作部分主要包括：电脑控制端、投影仪和物镜等。电脑控制端除了控制该系统未罗列出的一些精密移动机构外，主要用于产生光模块，如：虚拟传送带；投影仪将光模块映射到物镜的入口处；物镜将投影仪输出的光模块汇聚到ITO三明治机构中。
在用该装置实现细胞工厂的构建和精准发酵的过程中，1)用固定胶按图1所示准备ITO三明治机构；2)通过微纳3D打印技术在ITO三明治机构的下表面制造出细胞仓库和细胞培养板，并准备相应的细胞板架；3)将该ITO三明治机构放置在光操作机构上，调节光模块及ITO三明治机构的位置，使得将设计好的光模块能够映射到ITO三明治机构中；4)在ITO三明治机构的右端固 定胶的缝隙处注入适量的微生物细胞的细胞板架；5)借助光诱导介电泳力对微生物细胞进行操作和筛选，将筛选出的优良细胞移到细胞板架上；6)将装载着微生物细胞的细胞板架移动到虚拟传送带上，并用传送带将其传送到细胞仓库存储或者直接再次传送到细胞培养板中；7)直接在细胞培养板里面加入微发酵原料和改变发酵参数进行发酵参数优化，并且细胞培养板能够从ITO三明治机构的前端固定胶的缝隙处移出。
ITO三明治机构是该发明实施的基础，其结构如图2所示，制造过程简要地包括以下几个步骤：1)在25mm×25mm×1mm的透明玻璃基底上用磁控溅射的方法沉积一层厚为180nm左右的ITO层，构成了ITO玻璃；2)利用等离子体增强化学气相沉积方法，在ITO表面镀一层0.5-1μm厚的氢化非晶硅光电导层材料；3)利用3D微滴定装置将固定胶按设计好的结构滴加在氢化非晶硅表面，厚度为百微米级别，并将另一层ITO玻璃粘贴在固定胶的上表面。作为另一种优选方案，也能够直接用双面胶裁按设定的结构和尺寸，并粘贴在氢化非晶硅表面，而后将另一层ITO玻璃粘在双面胶另一面，制成ITO三明治机构。
细胞仓库用来存储筛选后并放置在细胞板上的微生物细胞，细胞仓库结构简图如图3所示。在该实施例中，一共有3层，每层有3格，每一格能够放置1片500μm×500μm细胞板架，层高为50μm左右。每格底部由一个宽度为30μm左右厚度为2μm左右的档条，用于放置细胞板架。作为优先方案，该细胞仓库是用3D打印技术将聚乙二醇二丙烯酸酯的水凝胶材料在氢化非晶硅表面从下往上逐层打印逐层固化制造而成的。
细胞板架用来装载微生物细胞，作为优选方案，细胞板架也是用3D打印技术和聚乙二醇二丙烯酸酯水凝胶材料制成。细胞板架有长度都为500μm，高度为7-10μm，宽度最长为500μm、逐渐减少50μm的多种规格，如图4所示给出了一种实施例。在该实施例中有三种规格的细胞板架，分别是500μm×500μm、500μm×450μm和500μm×400μm的规格。规格为500μm×500μm的细胞板架放置在最上层，500μm×450μm规格的放置在中间层，500μm×400μm规格的放置在最低层。并且所有细胞板架放置在细胞仓库上时，要使细胞板架贴到细胞仓 库的最里面，确保从下往上的光能够照射到每一层的细胞板架上，即，光诱导介电泳力能够作用到每一层的细胞板架。
虚拟传送带用于将细胞板架从一端送到另一端，其完全由PC端设置的光模块通过投影仪和物镜等映射到ITO三明治机构内。因为它不是实际存在的物理结构，而只是光学图像投影，因此，该发明中称为“虚拟传送带”。在一个实施例中，在PC机端按图5(A)设置光模块。该光模块主要由垂直的两条并排的正方形格子组成，两条垂直的光模块分别沿着其长度方向交替地运动着；在两条光模块的交叉位置额外设置了一个静态的方格，用于将细胞仓库内的细胞板架移动到垂直的两个光模块上。光模块方格的宽度L由PC机控制端的光模块与映射到ITO三明治机构内图案的缩小比值乘以虚拟传送带的实际需求尺寸决定。图5(B)是按图5(A)的光模式产生的虚拟传送带。两条光模块分别映射在ITO三明治机构的两个垂直缝隙中间，水平方向的传送带用于将细胞板架传送到细胞仓库，竖直方向的传送带用于将细胞仓库内的细胞板架传送到细胞培养板。传送带每个小方格的内的正方形宽度略大于500μm。
细胞工厂的整体结构如图6所示。在这个实施例中，ITO三明治机构的两条垂直的缝隙构成了细胞工厂的所有空间。在这个空间里，细胞仓库固定在氢化非晶硅表面前后向缝隙的后面，细胞工厂的右侧有一个进口和前侧有一个出口。进口主要用于输入细胞和细胞板架，并在进口的附近有一块空隙用于对微生物细胞进行筛选，借助于光诱导介电泳力按照微生物细胞的机械或生理特将性能良好的微生物细胞筛选出来，并移到细胞板架上表面。细胞仓库的前侧出口位置放置一个细胞培养板，细胞培养板能够用于直接培养微生物细胞，并能够取出其中的微生物细胞。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (9)

  1. 一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，包括：
    ITO三明治机构，包括第一ITO玻璃和第二ITO玻璃；所述的第一ITO玻璃包括第一玻璃基底和设置在第一玻璃基底表面的第一ITO薄膜层；所述的第二ITO玻璃包括第二玻璃基底、设置在第二玻璃基底上的第二ITO薄膜层和设置在第二ITO薄膜层上的氢化非晶硅层；所述氢化非晶硅层与第一ITO薄膜层之间形成中间层；所述中间层包括一个入口和一个出口；在第一ITO玻璃和第二ITO玻璃之间施加可变频率的交流电场；
    细胞工厂机构，所述的细胞工厂机构设置在所述中间层内，所述细胞工厂机构包括细胞仓库、虚拟传送带、细胞培养板和细胞板架，所述细胞板架通过虚拟传送带在中间层的入口、细胞仓库和细胞培养板之间移动；
    观测机构，用于观测或记录所述细胞工厂机构中的操作过程；
    光操作机构，用于在所述氢化非晶硅层上形成光模块，产生非均匀电场。
  2. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的中间层包括用于粘合所述氢化非晶硅层与第一ITO薄膜层的固定胶；所述固定胶形成一条通道，所述通道与中间层的入口和出口相连通，所述入口用于输入细胞或细胞板架，所述出口用于取出细胞或细胞培养板。
  3. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的细胞仓库包含若干单元格，所述单元格用于放置细胞板架，所述细胞板架在细胞仓库中放置时，从上往下，细胞板架的宽度依次减小。
  4. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的虚拟传送带由若干光模块构成，所述虚拟传送带包括用于在中间层入口与细胞仓库之间传送细胞板架的第一虚拟传送带、用于在细胞仓库与细胞培养板之间传送细胞板架的第二虚拟传送带和用于将细胞板架置 于细胞仓库或从细胞仓库中取出的第三虚拟传送带；所述的第一虚拟传送带和第二虚拟传送带在水平面上移动，所述的第三虚拟传送带在铅直面上移动。
  5. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的细胞培养板由尺寸相等的若干小方格排列形成。
  6. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的观测机构是位于所述ITO三明治机构上方的CCD。
  7. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的光操作机构包括电脑、投影仪和物镜，在电脑上产生特定形状的光模块，通过投影仪将光模块映射到物镜入口处，物镜将光模块汇聚到ITO三明治机构中。
  8. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的细胞仓库是用3D打印技术将聚乙二醇二丙烯酸酯的水凝胶材料在氢化非晶硅表面从下往上逐层打印逐层固化制造而成的。
  9. 根据权利要求1所述的一种基于光诱导介电泳技术构建的微生物细胞工厂，其特征在于，所述的细胞板架是用3D打印技术用聚乙二醇二丙烯酸酯水凝胶材料制成。
PCT/CN2019/114366 2019-01-16 2019-10-30 基于光诱导介电泳技术构建的微生物细胞工厂及其应用 Ceased WO2020147371A1 (zh)

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