Laboratory continuous evolution device and method

Abstract

The invention discloses a continuous evolution device for a laboratory. The continuous evolution device comprises a cell culture device; the feeding device is used for feeding and discharging the culture device; the biomass monitoring device comprises a first detection unit; measuring the part of the culture solution on line through a first detection unit so as to obtain biomass data in the culture solution; the fed-batch monitoring device comprises a second detection unit; the feeding amount or the discharging amount is measured on line, so that fed-batch amount data is obtained; and the controller is capable of collecting biomass data and fed-batch data and controlling the operation of the evolution device. According to the invention, the continuous evolution process of the laboratory can be effectively monitored, so that the continuous evolution efficiency of the laboratory is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a laboratory continuous evolution device and method. Background Art

[0002] With the advancement of bioinformatics and genetic engineering, it is becoming increasingly important to use the natural selection process to obtain and understand new microbial phenotypes. Therefore, laboratory evolution is a powerful technical method that can not only study the evolutionary forces that affect strain phenotype, performance and stability, but also obtain production strains containing beneficial mutations. In practice, laboratory evolution has been widely used in various aspects of microbial physiology related to industrial bioproduction, further demonstrating the importance of this technology as a biotechnological engineering method.

[0003] Generally speaking, laboratory evolution can be achieved through batch culture in shake flasks. That is, at regular intervals, aliquots of the culture are manually transferred to culture flasks filled with fresh culture medium for the next round of growth. Although this method has low requirements for experimental conditions, it is time-consuming and labor-intensive, and the strains obtained are unstable and prone to degeneration due to incomplete evolution. As an alternative to manual evolution, the use of bioreactors to build a chemostat can achieve continuous culture and evolution of cells. However, the conditions for achieving constant state are relatively harsh, not only requiring repeated exploration and adjustment of experimental parameters, but also changes in conditions as cells continue to evolve, which will further increase the risk of experimental failure. Summary of the invention

[0004] The goal of the present invention is to improve the success rate of laboratory evolution, especially to improve the simplicity and efficiency of evolution operation. On the one hand, the present invention realizes the miniaturization of cell culture devices and the continuity and automation of the operation process. On the other hand, it also designs stable online monitoring of the cell evolution state, and feeds back and adjusts the operation process by processing the monitoring data, so as to obtain the desired biological species (such as microbial strains) in a short time.

[0005] Therefore, one object of the present invention is to provide a laboratory continuous evolution device. Another object of the present invention is to provide a laboratory continuous evolution method.

[0006] The present invention provides a laboratory continuous evolution device, comprising:

[0007] a cell culture device for growing and evolving cells;

[0008] a feeding device for feeding and discharging materials from the culture device;

[0009] A biomass monitoring device comprises a first detection unit; the biomass monitoring device is configured to extract a portion of the culture fluid in the cell culture device and perform online measurement of the portion of the culture fluid through the first detection unit, thereby obtaining biomass data in the culture fluid;

[0010] A flow monitoring device, comprising a second detection unit; the flow monitoring device is configured to obtain flow amount data by online measuring the amount of feed or discharge;

[0011] A controller is provided, which can collect biomass data and feed rate data, and control the operation of the evolution device.

[0012] Furthermore, the biomass data and flow rate data obtained are used to analyze / evaluate the adaptability and / or evolutionary status of the cells.

[0013] Optionally, the second detection unit is an electronic balance for measuring the weight change of the feed bottle or the discharge bottle.

[0014] Furthermore, the flow addition monitoring device has a feed pipeline and a discharge pipeline, the feed pipeline includes a feed pump and a feed pipe, and the discharge pipeline includes a discharge pipe and a discharge pump; wherein, one end of the discharge pipe is located in the culture bottle, and the end of the discharge pipe located in the culture bottle is arranged to have a certain height from the bottom of the culture bottle; the feed pump and the discharge pump are respectively controlled by a controller, and are arranged so that, within a flow addition cycle, the workload of the discharge pump is greater than or equal to the workload of the feed pump.

[0015] Optionally, the feed monitoring device has two / multiple feed pumps, and the flow rate and / or running time of each feed can be set separately.

[0016] The present invention also provides a laboratory continuous evolution method, using the laboratory continuous evolution device of the present invention, the method comprising:

[0017] The first step is to set the parameters;

[0018] In the second step, after a certain interval, the stirrer stops stirring and the circulation pump starts circulating; after the circulation is completed, the first detection unit is used to measure the biomass data in the culture solution, and the time of data collection is recorded;

[0019] In the third step, if the biomass value detected by the first detection unit is less than the first biomass (OD_H), the stirrer restarts stirring, the evolution continues, and the second step is continued; if the biomass value detected by the first detection unit is not less than the first biomass, the fourth step is executed;

[0020] The fourth step is a feeding cycle: start the feed pump to feed, and continue for a certain feeding time, and start the discharge pump to discharge at the same time or later, and continue for a certain discharge time; when the feed pump and the discharge pump finish working, measure the flow rate data online through the second detection unit, and record the time of data collection; then the circulation pump starts working, and after the circulation ends, use the first detection unit to measure the biomass data in the culture solution online, and record the time of data collection, and enter the fifth step;

[0021] Step 5: If the biomass value detected by the first detection unit is not less than the second biomass (OD_L), repeat step 4 and continue a new cycle of feeding; if the biomass value detected by the first detection unit is less than the second biomass, suspend feeding, restart the agitator, continue the evolution, and execute step 2;

[0022] In the sixth step, during the evolution process, the collected biomass data and flow rate data are analyzed to evaluate the adaptability and / or evolutionary status of the cells.

[0023] The laboratory continuous evolution method of the present invention may further include: a seventh step, based on the analysis / evaluation results of the sixth step, changing the preset parameters and / or evolution conditions, and then proceeding to the second step.

[0024] Optionally, changing the evolution conditions may include changing the composition and / or ratio of the feed solution, and / or changing the culture environment in the incubator.

[0025] Optionally, changing the evolution condition is changing the ratio of the feed solution by adjusting the rotation speed ratio of two / more feed pumps, thereby changing the ratio between the various components of the feed solution.

[0026] Optionally, the analysis / evaluation method in step 6 includes one of the following two methods:

[0027] The first method is to reflect the degree of cell evolution by comparing the change in the growth time (Gt) required for each growth from the second biomass (OD_L) to the first biomass (OD_H) when the preset first biomass (OD_H) is greater than the second biomass (OD_L); at the same time, the degree of cell evolution can also be further reflected by the rate of change of the monitored flow rate;

[0028] The second method is to reflect the degree of cell evolution by calculating the rate of change of the flow rate when the preset first biomass (OD_H) is equal to the second biomass (OD_L).

[0029] As can be seen from the above scheme, a laboratory continuous evolution device of the present invention can realize the automatic control and continuous operation of laboratory evolution. In order to obtain accurate biomass data, the biomass can be monitored online in an external circulation manner. Further, the stability of monitoring is increased by the cooperation of the control system. Since the present invention can obtain data such as the biomass and flow addition amount of cells in the continuous evolution process online, and after being processed by the processor according to the set parameters and evolution method, the automatic operation of the culture device and the flow addition device is controlled by the controller module, the laboratory continuous evolution process can be effectively monitored, thereby improving the efficiency of laboratory continuous evolution. Therefore, through the present invention, the process of continuous evolution in the laboratory can be easily realized, and the evolution process can be effectively monitored, thereby improving the efficiency of laboratory continuous evolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the composition of a laboratory continuous evolution device in one embodiment of the present invention.

[0031] Figure 2 It is a schematic diagram of the internal modules of a controller in one embodiment of the present invention.

[0032] Figure 3 It is a flow chart of implementing the laboratory continuous evolution method of the present invention using the laboratory continuous evolution device of the present invention.

[0033] 1-incubator, 2-culture bottle, 3-magnetic stirrer, 4-feed bottle, 5-feed pump, 6-discharge bottle, 7-discharge pump, 8-first detection unit, 9-circulation pump, 10-second detection unit, 11-controller, 12-display screen, 13-knob, 14-magnetic rotor, 15-air filter. DETAILED DESCRIPTION

[0034] The laboratory continuous evolution device described in the present invention is composed of a cell culture device, a flow addition device, a biomass monitoring device, a flow addition monitoring device and a controller. The present invention also discloses a laboratory continuous evolution method, which can effectively monitor the continuous evolution process, thereby improving the efficiency of continuous evolution.

[0035] The specific implementation of the present invention is further described in detail below in conjunction with the drawings in the specification. The drawings are only for illustrative purposes and represent only schematic diagrams rather than physical drawings, and cannot be understood as limiting the present patent; in order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0036] Except for special markings, the same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or component referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limitations on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0037] "Laboratory evolution" refers to setting expected conditions in the laboratory, simulating the process of biological evolution through the continuous adaptation of organisms to the living environment, and observing the phenomenon of biological evolution. It is particularly suitable for microbial research, such as studying the evolutionary potential that affects the phenotype, performance and stability of strains, and quickly obtaining industrial production strains containing beneficial mutations, for example, by implementing the natural selection rules proposed in Darwin's theory on the laboratory bench to produce evolved microbial strains with desired characteristics.

[0038] The present invention is different from the "manual" and "batch" laboratory evolution culture in the prior art, but can realize a "continuous" evolution process, so it is called "laboratory continuous evolution".

[0039] A laboratory continuous evolution device of the present invention comprises:

[0040] a cell culture device for growing and evolving cells;

[0041] a feeding device for feeding and discharging materials from the culture device;

[0042] A biomass monitoring device comprises a first detection unit; the biomass monitoring device is configured to extract a portion of the culture fluid in the cell culture device and perform online measurement of the portion of the culture fluid through the first detection unit, thereby obtaining biomass data in the culture fluid;

[0043] A flow monitoring device, comprising a second detection unit; the flow monitoring device is configured to obtain flow amount data by online measuring the amount of feed or discharge;

[0044] A controller is provided, which can collect biomass data and feed rate data, and control the operation of the evolution device.

[0045] like Figure 1As shown, in one embodiment, the cell culture device of the present invention may optionally include: an incubator 1, especially an incubator capable of controlling the temperature of the culture medium; a culture bottle 2 for containing the cell culture medium; and a stirrer 3 for stirring the culture medium.

[0046] Optionally, the incubator 1 capable of controlling the temperature of the culture solution includes but is not limited to a constant temperature incubator, a constant temperature and humidity incubator, a carbon dioxide incubator, and the like.

[0047] Optionally, the culture bottle 2 for containing the cell culture fluid is a commonly used cell culture container in the laboratory, including but not limited to a shake flask, a screw-mouth bottle, etc., and the material includes but is not limited to plastic, glass, stainless steel, etc.

[0048] Optionally, the stirrer 3 for stirring the culture solution includes but is not limited to a magnetic stirrer, and an oscillator that can also achieve a stirring function.

[0049] Further optionally, the oxygen concentration in the culture solution can be changed by some methods, including but not limited to installing an air filter 15 on the culture bottle, and flushing air, oxygen, nitrogen, carbon dioxide gas, etc. into the culture bottle or incubator.

[0050] The feed addition device of the present invention may optionally include: a feed bottle 4 and a feed pump 5 for the inflow of culture solution; and a discharge bottle 6 and a discharge pump 7 for the outflow of culture solution.

[0051] The feed bottle 4 and the discharge bottle 6 can be commonly used liquid containers in laboratories, including but not limited to shake bottles, screw-mouth bottles, etc., and the materials include but are not limited to plastic, glass, stainless steel, etc.

[0052] The feed pump 5 for controlling the addition of culture solution and the discharge pump 7 for controlling the outflow of culture solution may be peristaltic pumps, including but not limited to DC motor peristaltic pumps, stepper motor peristaltic pumps, etc. The feed pump 5 and the discharge pump 7 preferably have an external control interface, so as to be controlled by the controller 11.

[0053] Furthermore, if the culture solution is easy to stratify, a stirrer can be installed at the bottom of the feed bottle 4. Optionally, in order to reduce the risk of contamination of the culture solution in the feed bottle during long-term continuous evolution, the feed bottle 4 can be cooled. The cooling method includes but is not limited to placing the feed bottle 4 in an ice box.

[0054] Further optionally, two or more feed bottles 4 and feed pumps 5 can be configured in the flow addition device. By adjusting the speed ratio between the two / multiple feed pumps 5, changes in the composition of the feed liquid (culture liquid), such as gradient changes, such as gradient increases, can be achieved.

[0055] In the present invention, the feed pump 5 and the discharge pump 7 are two independent pumps, which are controlled by controllers respectively, so that the rotation speed (flow rate) and / or running time of the sample feeding / discharging can be set respectively.

[0056] When there are two or more feed pumps, they are also controlled by the controller separately, so that the rotation speed (flow rate) and / or running time of each injection can be set separately.

[0057] In one embodiment, the speed (flow rate) of the feed pump / discharge pump is manually set, and the running time of each time is a parameter preset by the controller. In actual operation, it is not limited to this.

[0058] The biomass monitoring device of the present invention contains a first detection unit 8, through which part of the culture solution is measured to obtain biomass data in the culture solution. The "biomass data" in the present invention particularly refers to the number of cells or cell weight per unit liquid volume. For example, the biomass can be characterized by an OD value, or by a microscopic counting method. Therefore, the first detection unit 8 can be selected as a cell turbidity meter or a spectrophotometer, or can be selected as other detection equipment that can reflect the biomass, including but not limited to a flow cytometer, a microscope, etc. The first detection unit 8 preferably has an external communication interface to facilitate communication with the controller 11.

[0059] In addition, the biomass monitoring device may further include a circulation pump 9 for circulating the culture solution. The circulation pump 9 for circulating the culture solution may be a peristaltic pump, including but not limited to a DC motor peristaltic pump, a stepper motor peristaltic pump, etc. The circulation pump 9 preferably has an external control interface so as to be controlled by the controller 11.

[0060] During the cyclic measurement process, a portion of the culture liquid in the culture bottle 2 is pumped out through the circulation pump 9 , has its biomass data measured by the first detection unit 8 , and then returns to the culture bottle 2 .

[0061] The flow-addition monitoring device of the present invention comprises a second detection unit 10; the flow-addition monitoring device obtains flow-addition amount data by online measuring the amount of feed or discharge. The "flow-addition amount data" in the present invention refers to the amount of liquid added by flow, especially the volume or weight of liquid fed into the cell culture device by the flow-addition device, or the volume or weight of liquid discharged in a feeding cycle. For example, the second detection unit 10 is an electronic balance. The second detection unit 10 is preferably provided with an external communication interface to facilitate communication with the controller 11. The second detection unit 10 can be configured to measure the weight change of the feed bottle 4, or it can be configured to measure the weight change of the feed bottle 6. That is, the flow addition amount of the culture solution can be monitored by measuring the weight change of the feed bottle 4, or it can be monitored by monitoring the weight change of the discharge bottle 6.

[0062] In particular, when the culture solution in the feed bottle 4 needs to be stirred or placed in an ice box, which is inconvenient for weighing, it is preferred to monitor the feed amount by the weight change of the discharge bottle 6. When the cell culture temperature is high and the culture solution in the culture bottle 2 evaporates more, it is preferred to monitor the feed amount by the weight change of the feed bottle 4.

[0063] Through the second detection unit 10, reliable flow rate data (volume or weight) can be obtained, which can be used to analyze / evaluate the adaptability and / or evolution status of cells.

[0064] Liquid pipeline. Feed pipeline: The culture fluid feed bottle is connected to the feed pipe inserted on the top of the cell culture bottle through the feed pump. Discharge pipeline: The culture fluid discharge bottle is connected to the discharge pipe inserted on the top of the cell culture bottle through the discharge pump. Circulation pipeline: The first detection unit, the circulation pump, the circulation outlet pipe and the circulation inlet pipe connected to the cell culture bottle form a circulation pipeline.

[0065] It should be noted that the rotation direction of the feed pump should be set to the direction in which the culture fluid flows into the cell culture bottle, and the rotation direction of the discharge pump should be set to the direction in which the culture fluid flows out of the cell culture bottle.

[0066] The end of the discharge pipe in the cell culture bottle is set to have a certain height from the bottom of the culture bottle, that is, it is set to the height of the liquid level with a certain amount of liquid in the culture bottle. In this way, after the discharge pump has been pumping for a certain period of time, once the liquid level in the bottle drops below the end of the discharge pipe, it will no longer be pumped out. Thereby maintaining a certain volume of liquid in the bottle, and at this time, the end of the discharge pipe just touches the liquid level in the cell culture bottle. The end of the circulating liquid outlet pipe should be set at a position closer to the bottom of the bottle than the discharge pipe, that is, the circulating liquid outlet pipe should go deep below the liquid level in the cell culture bottle.

[0067] The controller of the present invention can collect biomass data and flow rate data, and control the operation of the evolution device. Figure 2 As shown, in one embodiment, the controller 11 includes: a data acquisition module for collecting data; a data storage module for storing data; a processor module for processing data; a control module for controlling the operation of other device components, such as controlling the switch and / or speed of the agitator 3, the feed pump 5, the discharge pump 7 and the circulation pump 9. In addition, an external communication module for communicating with the outside may be optionally included. Several modules for interacting with the operator may also be optionally included, such as a display module for displaying data, and / or a parameter setting module for parameter setting. An equipment start-stop module for controlling the start and stop of the system, and / or a power supply module may also be optionally included.

[0068] Furthermore, the data collection module may include: a biomass collection module for collecting cell growth data; and a weight collection module for collecting the amount of culture fluid added.

[0069] Furthermore, the control module may include: an agitator control module for controlling the stirring of the culture solution; a circulation pump control module for controlling the circulation of the culture solution; a feed pump control module for controlling the flow of the culture solution from the feed bottle 4 into the cell culture bottle 2; and a discharge pump control module for controlling the flow of the culture solution from the cell culture bottle 2 to the discharge bottle 6.

[0070] Further optionally, the controller is connected to an external computer. For example, the collected data is first stored in the controller and then can be output to the external computer. For example, the controller can accept parameters and specifications input from the external computer. For example, the modification and upgrade of the continuous evolution implementation method in the storage module also needs to be carried out after connecting to the external computer.

[0071] The laboratory continuous evolution device described in the present invention is configured to execute the laboratory continuous evolution method described in the present invention.

[0072] The laboratory continuous evolution method of the present invention comprises:

[0073] The first step is to set parameters. The preset parameters may include but are not limited to one or more of the following: first biomass (OD_H), second biomass (OD_L), interval time (Pt), cycle time (Ct), feed time (INt), discharge time (OUTt), etc. Generally speaking, the first biomass (OD_H) is set to be greater than or equal to the second biomass (OD_L). Generally speaking, the discharge time (OUTt) is set to be greater than or equal to the feed time (INt).

[0074] In the second step, after a certain interval (Pt), the agitator stops stirring and the circulation pump 9 starts circulating, and the circulation time is Ct. After reaching a certain circulation time Ct, the circulation pump 9 stops running (i.e., the circulation ends). After a certain circulation time Ct, the biomass in this part of the culture solution in the circulation monitoring system can be considered to be consistent with the biomass of the culture solution in the cell culture device. After the circulation ends, the first detection unit 8 is used to measure the biomass data in the culture solution online, and the time of data acquisition is recorded.

[0075] In the third step, if the biomass value detected by the first detection unit 8 is less than the first biomass (OD_H), the stirrer restarts stirring, the evolution continues, and the second step is continued. If the biomass value detected by the first detection unit 8 is not less than the first biomass (OD_H), the fourth step is executed.

[0076] The fourth step is a feeding cycle. Start the feed pump 5 for feeding, and the duration of the operation of the feed pump 5 is the feeding duration (INt). Optionally, the discharging can be started at the same time as the feeding starts, or the discharging can be started after waiting for a waiting time (Wt) after the feeding starts. The discharging is started by starting the discharging pump 7, and the duration of the operation of the discharging pump 7 is the discharging duration (OUTt). When the feed pump 5 and the discharging pump 7 have finished working, the flow addition data is measured online by the second detection unit 10, and the time of data collection is recorded. Then the circulating pump 9 starts working, and the cycle duration is Ct. After the cycle ends, the first detection unit 8 is used to measure the biomass data in the culture solution online, and the time of data collection is recorded, and the fifth step is entered.

[0077] It should be noted that in the present invention, the volume of the culture fluid in the cell culture bottle is actually determined by the height of the end of the discharge pipe from the bottom of the culture bottle. Generally speaking, the workload of the discharge pump is set to be greater than or equal to the workload of the feed pump, so that the culture fluid is extracted to below the end of the discharge pipe each time, thereby keeping the volume of the culture fluid in the culture bottle relatively fixed. The phenomenon of continuous accumulation and increase of liquid in the culture bottle will not occur. In specific operations, this can be achieved by adjusting the rotation speed and working time (INt and OUTt) of the feed pump and the discharge pump. For example, when the diameter and rotation speed of the feed peristaltic pump and the discharge peristaltic pump are the same, the discharge time OUTt is set to be greater than or equal to the feed time INt.

[0078] In the fifth step, if the biomass value detected by the first detection unit 8 is not less than the second biomass (OD_H), the fourth step is repeated to continue a new cycle (a new round) of feeding. If the biomass value detected by the first detection unit 8 is less than the second biomass (OD_H), the feeding is suspended, the stirrer restarts stirring, the evolution continues, and the second step is executed.

[0079] In the sixth step, during the evolution process, the collected biomass data and flow rate data are analyzed to evaluate the adaptability and / or evolutionary status of the cells.

[0080] Specifically, there are two possible analysis / evaluation methods for step 6:

[0081] The first one is that when the preset first biomass (OD_H) is greater than the second biomass (OD_L), the degree of evolution of the cells is reflected by comparing the changes in the growth time (Gt) required for each growth from the second biomass (OD_L) to the first biomass (OD_H). Generally speaking, the shorter the required growth time (Gt), the better the adaptability of the cells to the culture conditions and the more fully evolved they are. At the same time, the degree of evolution of the cells can also be further reflected by calculating the rate of change of the flow addition amount. The "rate of change of the flow addition amount" refers to the ratio of the cumulative change in the total amount of feed / discharge obtained after one or more feeding cycles to the time used. For example, at the beginning of evolution, after several (a certain number, such as 5) feeding cycles, the cumulative feed amount changes from 50 grams to 100 grams, and the time used is 6 hours, that is, the "rate of change of the flow addition amount" is 50 grams / 6 hours. As the evolution progresses, after several (a certain number, such as 5) feeding cycles, the cumulative feed amount changes from 200 grams to 250 grams, and the time used is 4 hours, that is, the "rate of change of feed amount" is 50 grams / 4 hours. This shows that the adaptability of cells to the culture conditions is getting better and better. Generally speaking, the faster the rate of change of feed amount, the better the adaptability of cells to the culture conditions and the more fully evolved.

[0082] The second type is that when the preset first biomass (OD_H) is equal to the second biomass (OD_L), the evolution degree of the cell is reflected by calculating the rate of change of the flow addition amount. The "rate of change of the flow addition amount" refers to the ratio of the change of the total amount of feed / discharge accumulated after one or more feeding cycles to the time used. For example, at the beginning of evolution, after several (a certain number, such as 5) feeding cycles, the cumulative feed amount changes from 50 grams to 100 grams, and the time used is 6 hours, that is, the "rate of change of the flow addition amount" is 50 grams / 6 hours. As the evolution proceeds, after several (a certain number, such as 5) feeding cycles, the cumulative feed amount changes from 200 grams to 250 grams, and the time used is 4 hours, that is, the "rate of change of the flow addition amount" is 50 grams / 4 hours. It shows that the adaptability of cells to culture conditions is getting better and better. Generally speaking, the rate of change of the flow addition amount is getting larger and larger, indicating that the adaptability of cells to culture conditions is getting better and better, and the evolution is more sufficient.

[0083] Optionally, the laboratory continuous evolution method of the present invention may also include:

[0084] Step 7. Based on the results of the analysis / evaluation in step 6, change the preset parameters and / or evolution conditions, and then proceed to step 2.

[0085] For example, the changed preset parameters may include but are not limited to one or more of the following: the first biomass (OD_H), the second biomass (OD_L), the interval time (Pt), the feeding time (INt), the discharging time (OUTt), the speed (flow rate) of the feeding pump, the speed (flow rate) of the discharging pump, etc.

[0086] For example, the changing of the evolution conditions may be changing the composition and / or ratio of the feed solution, and / or changing the culture environment in the incubator, etc.

[0087] For example, in a device having two / more feed pumps 5 , the ratio of the various components of the feed solution can be changed by adjusting the rotation speed ratio of the two / more feed pumps 5 .

[0088] As biological evolution (e.g., microbial evolution) proceeds, the state of cell growth may be too good, resulting in too much total amount of flow required to decrease from the first biomass (OD_H) to the second biomass (OD_L) each time, or the growth time (Gt) required to grow from the second biomass (OD_L) to the first biomass (OD_H) each time is too short. At this time, it is necessary to adjust some parameters or experimental conditions (such as feed solution and / or culture environment) to better promote / monitor laboratory evolution.

[0089] At the other extreme, the cell growth state is too poor, resulting in a decrease in the amount of flow required each time from the first biomass (OD_H) to the second biomass (OD_L), or the growth time (Gt) required each time from the second biomass (OD_L) to the first biomass (OD_H) is too long. At this time, it is necessary to adjust some parameters or experimental conditions (such as feed solution and / or culture environment) to better promote / detect laboratory evolution.

[0090] Through the method of the present invention, technicians in this field can easily achieve controllable and detectable continuous evolution in the laboratory, thereby directionally controlling the evolutionary direction of microorganisms and obtaining target microbial strains, such as production strains that meet specific needs (such as industrial use). Specific embodiment 1:

[0092] This embodiment provides a laboratory continuous evolution device. Figure 1 Use the connection method in to connect the hardware devices in the laboratory continuous evolution device.

[0093] Among them, the cell culture bottle, magnetic stirrer, circulating peristaltic pump and cell turbidity meter are placed inside the constant temperature incubator, and the external control interfaces of the magnetic stirrer and circulating peristaltic pump are respectively connected to the stirrer control module and circulating pump control module of the controller, and the data interface of the cell turbidity meter is connected to the biomass collection module of the controller. In addition, the feed bottle, the discharge bottle, the feed peristaltic pump, the discharge peristaltic pump and the electronic balance for obtaining the flow addition amount of the culture solution are placed outside the constant temperature incubator, and the external control interfaces of the feed peristaltic pump and the discharge peristaltic pump are respectively connected to the feed pump control module and the discharge pump control module of the controller, and the data interface of the electronic balance is connected to the flow addition amount collection module of the controller.

[0094] It should be noted that when the volume of the constant temperature incubator is relatively small, the circulating peristaltic pump and the cell turbidity meter can also be placed outside the constant temperature incubator.

[0095] At the same time, in order to increase the oxygen content in the culture bottle during the cell growth process, a pipeline with an air filter device is installed on the top of the culture bottle in this embodiment.

[0096] Taking the evolution experiment of Escherichia coli resistance to ampicillin as an example, the culture medium content in the culture bottle is 50 ml, the culture medium is LB liquid culture medium containing ampicillin (each liter of LB liquid culture medium contains 10 grams of peptone, 5 grams of yeast powder, 10 grams of sodium chloride, and the pH is adjusted to 7.2 with sodium hydroxide or dilute hydrochloric acid, and high-pressure sterilization is performed for 30 minutes), and the initial concentration of ampicillin is 1 μg / ml.

[0097] In this embodiment, the silicone tubes used in all liquid pipelines have an inner diameter of 2 mm and an outer diameter of 4 mm; the speed of the magnetic stirrer is set to 200 rpm; the speed of the circulating peristaltic pump is set to 100 ml / min. The culture medium in a feed bottle is LB liquid culture medium containing 1 μg / ml ampicillin, and the corresponding initial speed of the feed peristaltic pump is set to 50 ml / min. The speed of the discharge peristaltic pump is set to 50 ml / min.

[0098] Optionally, a second feed bottle is also provided, the culture solution in the second feed bottle is LB liquid culture medium containing 100 μg / ml ampicillin, and the initial speed of the corresponding feed peristaltic pump is set to 0 ml / min.

[0099] This embodiment follows the Figure 2The modules inside the controller are connected by the connection method in . Among them, the parameter setting module is connected to the data storage module, and the set parameters will be stored in the storage module. The parameter setting is performed through the knob installed on the controller. The storage module is connected to the data acquisition module with an RS232 to TTL interface to obtain biomass data and flow addition data. The storage module also stores the pre-set continuous evolution implementation method. The processor module obtains the data in the data storage module and processes the data according to the stored implementation method and parameters. According to the results of data processing, the processor controls the operating status of the magnetic stirrer, the circulating peristaltic pump, and the feed peristaltic pump and the discharge peristaltic pump respectively by changing the state of the electromagnetic relay in the control module. The operating parameters and the data collected during the operation can be displayed through the LCD display module.

[0100] It is worth mentioning that the storage module can be connected to an external computer through the USB interface on the external communication module. At this time, the setting of parameters can be performed through the external computer, and the collected data can also be output to the external computer through the external communication module. In addition, the change and upgrade of the continuous evolution implementation method in the storage module also need to be carried out after connecting to the external computer. The equipment start-stop module controls the start and stop of the entire system by connecting to the processor module. Specific embodiment 2:

[0102] The present invention provides a method for continuous laboratory evolution. Figure 3 This method will be described.

[0103] Figure 3 It is a flow chart of the laboratory continuous evolution method of an embodiment of the present invention.

[0104] Taking the evolution experiment of Escherichia coli resistance to ampicillin as an example, first set the second biomass (OD_L) to 0.5, the first biomass (OD_H) to 1.5, the interval time (Pt) to 10 minutes, the cycle time (Ct) to 15 seconds, the feeding time (INt) to 10 seconds, and the discharging time (OUTt) to 20 seconds.

[0105] After the parameter setting is completed, the E. coli cultured overnight is inoculated into the culture bottle with an inoculum size of 0.1OD. Press the start button on the controller panel, and the system starts running and the agitator starts working. After the system runs for 10 minutes, the agitator stops stirring and the circulation pump starts circulating, with a cycle time of 15 seconds. After the cycle is completed, the biomass in the culture solution is measured online by the cell turbidity meter, and the data is sent to the biomass acquisition module of the controller. The acquisition module sends the biomass value and the acquisition time to the data storage module at the same time.

[0106] The processor obtains the data in the data storage module and compares the relationship between the currently measured biomass value and the set first biomass (OD_H). When the biomass value in the culture solution measured online is less than 1.5, the agitator restarts the stirring, and the cell culture continues. Repeat the above operation after the system runs for 10 minutes. When the biomass value in the culture solution measured online is greater than 1.5, it indicates that the system has completed a round of cultivation. At this time, the feed pump starts feeding, the feeding time is 10 seconds, and the discharge pump starts discharging, and the discharging time is 20 seconds. The electronic balance measures the flow addition of the culture solution online and sends the data to the flow addition acquisition module of the controller, and the acquisition module sends the value of the flow addition and the time of acquisition to the data storage module at the same time. Then the circulation pump starts working, the cycle time is 15 seconds, and after the cycle ends, the cell turbidity meter measures the biomass in the culture solution online, and sends the data to the biomass acquisition module of the controller, and the acquisition module sends the value of the biomass and the time of acquisition to the data storage module at the same time. The processor obtains the data in the data storage module and compares the relationship between the currently measured biomass value and the set second biomass (OD_L). When the biomass value in the culture solution measured online is greater than 0.5, the above process is repeated until the biomass value in the culture solution measured online is less than the second biomass value 0.5. At this time, the system will start a new round of evolution work.

[0107] As the evolution proceeds, the time required for each cell to grow from the second biomass (OD_L) to the first biomass (OD_H) gradually shortens, and the rate of change of the flow rate increases. At this time, the concentration of ampicillin in the feed culture solution is adjusted, and the evolution continues. For example, by manually replacing the feed culture solution, or by adjusting the speed ratio of the two feed peristaltic pumps, the concentration of ampicillin in the mixed feed culture solution is increased to 10 micrograms / ml, and then the evolution continues.

[0108] It is worth mentioning that as the concentration of antibiotics in the culture medium continues to increase, when cells cannot grow for a long time (such as the biomass data and flow rate data monitored online have no change for more than 24 hours), it means that the increase in antibiotics is too large. At this time, the concentration of antibiotics in the mixed feed culture medium can be reduced to allow the cells to resume growth and continue to evolve. For example, the concentration of ampicillin in the feed culture medium can be reduced by manually replacing the feed culture medium or by adjusting the speed ratio of the two feed peristaltic pumps.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents. These modifications or replacements are

[0110] The essence of the corresponding technical solution does not deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. A laboratory continuous evolution device, comprising: a cell culture device for growing and evolving cells; a feeding device for feeding and discharging materials from the culture device; A biomass monitoring device comprises a first detection unit; the biomass monitoring device is configured to extract a portion of the culture fluid in the cell culture device and perform online measurement of the portion of the culture fluid through the first detection unit, thereby obtaining biomass data in the culture fluid; A flow monitoring device, comprising a second detection unit; the flow monitoring device is configured to obtain flow amount data by online measuring the amount of feed or discharge; A controller is provided, which can collect biomass data and feed rate data, and control the operation of the evolution device.

2. The laboratory evolution device according to claim 1, wherein: The biomass data and flow rate data obtained are used to analyze / evaluate the adaptability and / or evolutionary status of the cells.

3. The laboratory evolution device according to claim 1, wherein: The second detection unit is an electronic balance, which is used to measure the weight change of the feeding bottle or the discharging bottle.

4. The laboratory evolution device as described in claim 1, wherein the flow-addition monitoring device has a feed pipeline and a discharge pipeline, the feed pipeline includes a feed pump and a feed pipe, and the discharge pipeline includes a discharge pipe and a discharge pump; wherein, One end of the discharge pipe is located in the culture bottle, and the end of the discharge pipe located in the culture bottle is set to have a certain height from the bottom of the culture bottle; the feed pump and the discharge pump are respectively controlled by a controller and are set so that, within a flow addition cycle, the workload of the discharge pump is greater than or equal to the workload of the feed pump.

5. The laboratory evolution device as described in claim 1, wherein the flow addition monitoring device has two / multiple feed pumps, and the flow rate and / or running time of each feed can be set separately.

6. A laboratory continuous evolution method, using the laboratory continuous evolution device described in any one of claims 1 to 5, the method comprising: The first step is to set the parameters; In the second step, after a certain interval, the agitator stops stirring and the circulation pump starts circulating; After the cycle is completed, the biomass data in the culture solution is measured using the first detection unit, and the time of data collection is recorded; In the third step, if the biomass value detected by the first detection unit is less than the first biomass (OD_H), the stirrer restarts stirring, the evolution continues, and the second step is continued; if the biomass value detected by the first detection unit is not less than the first biomass, the fourth step is executed; The fourth step is a feeding cycle: start the feed pump to feed, and continue for a certain feeding time, and start the discharge pump to discharge at the same time or later, and continue for a certain discharge time; when the feed pump and the discharge pump finish working, measure the flow rate data online through the second detection unit, and record the time of data collection; then the circulation pump starts working, and after the circulation ends, use the first detection unit to measure the biomass data in the culture solution online, and record the time of data collection, and enter the fifth step; Step 5: If the biomass value detected by the first detection unit is not less than the second biomass (OD_L), repeat step 4 and continue a new cycle of feeding; if the biomass value detected by the first detection unit is less than the second biomass, suspend feeding, restart the agitator, continue the evolution, and execute step 2; In the sixth step, during the evolution process, the collected biomass data and flow rate data are analyzed to evaluate the adaptability and / or evolutionary status of the cells.

7. The laboratory continuous evolution method as described in claim 6 further comprises Step 7. Based on the results of the analysis / evaluation in step 6, change the preset parameters and / or evolution conditions, and then proceed to step 2.

8. The laboratory continuous evolution method according to claim 7, wherein changing the evolution conditions may be changing the composition and / or proportion of the feed solution, and / or changing the culture environment in the incubator.

9. The laboratory continuous evolution method according to claim 8, wherein: Changing the evolution condition means changing the ratio of the feed solution, which is achieved by adjusting the speed ratio of two / more feed pumps, thereby changing the ratio between the various components of the feed solution.

10. The laboratory continuous evolution method according to claim 6, wherein the analysis / evaluation method in the sixth step comprises one of the following two: The first method is to reflect the degree of cell evolution by comparing the change in the growth time (Gt) required for each growth from the second biomass (OD_L) to the first biomass (OD_H) when the preset first biomass (OD_H) is greater than the second biomass (OD_L); at the same time, the degree of cell evolution can also be further reflected by the rate of change of the monitored flow rate; The second method is to reflect the degree of cell evolution by calculating the rate of change of the flow rate when the preset first biomass (OD_H) is equal to the second biomass (OD_L).

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