CN119101601A - A laboratory continuous evolution device and method - Google Patents

Abstract

The invention discloses a continuous evolution device in a laboratory, which comprises a cell culture device, a feeding device, a biomass monitoring device, a feeding monitoring device and a controller, wherein the feeding device is used for feeding and discharging the culture device, the biomass monitoring device comprises a first detection unit and is used for carrying out online measurement on a part of culture solution through the first detection unit so as to obtain biomass data in the culture solution, the feeding monitoring device comprises a second detection unit and is used for carrying out online measurement on the feeding or discharging amount so as to obtain feeding amount data, and the controller is used for collecting the biomass data and the feeding amount data and controlling the operation of the evolution device. The realization of the invention can effectively monitor the continuous evolution process of the laboratory, thereby improving the continuous evolution efficiency of the laboratory.

Description

Continuous evolution device and method for laboratory

Technical Field

The invention relates to the technical field of biology, in particular to a continuous evolution device and method for a laboratory.

Background

With advances in bioinformatics and genetic engineering, it is becoming increasingly important to utilize natural selection processes to obtain and understand new microbial phenotypes. Therefore, laboratory evolution is a powerful technical approach, which allows both the study of evolutionary forces affecting the phenotype, performance and stability of strains and the acquisition of production strains containing beneficial mutations. In practice, laboratory evolution has been widely applied to various aspects of microbial physiology associated with industrial bio-production, more demonstrating the importance of this technology as a method of biotechnology engineering.

In general, laboratory evolution can be achieved by batch culture in shake flasks. I.e., at regular intervals, aliquots of the culture were manually transferred to flasks with fresh medium for the next round of growth. Although the implementation mode has lower requirements on experimental conditions, the implementation mode is time-consuming and labor-consuming, and the obtained strain is unstable due to insufficient evolution, so that the degradation phenomenon is easy to occur. As an alternative method for manual evolution, continuous culture and evolution of cells can be realized by constructing a chemostat by using a bioreactor. However, the conditions for realizing the chemostat are severe, so that not only are repeated fumbling and adjustment of experimental parameters required, but also the risk of experimental failure is further increased due to the change of the conditions caused by the continuous evolution of cells.

Disclosure of Invention

The invention aims to improve the success rate of laboratory evolution, in particular to improve the simplicity and the efficiency of evolution operation. The invention realizes miniaturization of the cell culture device and continuous and automatic operation process, and designs stable on-line monitoring of the cell evolution state, and the operation process is fed back and adjusted by processing the monitoring data, so that the desired biological species (such as microorganism strain) is obtained in a short time.

To this end, an object of the present invention is to provide a laboratory continuous evolution device. It is another object of the present invention to provide a method of continuous evolution in a laboratory.

The invention provides a continuous evolution device in a laboratory, which comprises the following components:

a cell culture device for culturing and evolving cells;

A feeding device for feeding and discharging the culture device;

The biomass monitoring device is arranged to extract a part of the culture solution in the cell culture device and to perform online measurement on the part of the culture solution through the first detection unit so as to obtain biomass data in the culture solution;

the feeding monitoring device is arranged to obtain feeding amount data by measuring the feeding amount or the discharging amount on line;

a controller capable of collecting biomass data and feed-through data and controlling the operation of the evolution device.

Further, the biomass data and the fed-batch data it obtained are used to analyze/evaluate the state of adaptation and/or evolution of the cells.

Optionally, the second detection unit is an electronic balance for measuring a weight change of the feeding bottle or the discharging bottle.

Further, the feeding monitoring device is provided with a feeding pipeline and a discharging pipeline, the feeding pipeline comprises a feeding pump and a feeding pipe, and the discharging pipeline comprises a discharging pipe and a discharging pump; one end of the discharging pipe is positioned in the culture bottle, the tail end of the discharging pipe positioned in the culture bottle is arranged to be at a certain height from the bottle bottom of the culture bottle, and the feeding pump and the discharging pump are respectively controlled by the controller and are arranged so that the workload of the discharging pump is larger than or equal to that of the feeding pump in one feeding period.

Optionally, the feeding monitoring device is provided with two/more feeding pumps, and the flow rate and/or the running time of each feeding can be respectively set.

The invention also provides a laboratory continuous evolution method, which comprises the following steps of:

Firstly, setting parameters;

The second step, after a certain interval time, the stirrer stops stirring, the circulating pump starts circulating, after the circulation is finished, the first detection unit is used for measuring biomass data in the culture solution, and the time for data acquisition is recorded;

a third step of restarting stirring by the stirrer if the biomass value detected by the first detection unit is smaller than the first biomass (OD_H), continuing evolution, and continuing to execute the second step;

Starting a feeding pump to feed for a certain feeding time period, starting a discharging pump to discharge at the same time or later, and continuously discharging for a certain discharging time period, after the feeding pump and the discharging pump are finished working, measuring fed-batch data on line by a second detection unit, and recording the time of data acquisition;

a fifth step of repeating the fourth step to continue feeding for a new period if the biomass value detected by the first detecting unit is not less than the second biomass (OD_L), and suspending feeding if the biomass value detected by the first detecting unit is less than the second biomass, restarting stirring by the stirrer to continue evolution, and executing the second step;

and sixthly, analyzing the collected biomass data and fed-batch data in the evolution process, and evaluating the adaptability and/or the evolution state of the cells.

The laboratory continuous evolution method of the present invention may further comprise a seventh step of modifying the preset parameters and/or the evolution conditions based on the result of the analysis/evaluation of the sixth step, and then performing the second step.

Optionally, wherein altering the evolution condition may be altering the composition and/or ratio of the feed solution, and/or altering the culture environment within the incubator.

Alternatively, altering the evolution conditions is altering the feed solution ratio by adjusting the speed ratio of the two/more feed pumps, thereby altering the ratio between the individual components of the feed solution.

Optionally, the analysis/evaluation method of the sixth step includes one of the following two:

First, when the preset first biomass (OD_H) is larger than the second biomass (OD_L), the evolution degree of the cells is reflected by comparing the change of the growth time (Gt) required for each growth from the second biomass (OD_L) to the first biomass (OD_H);

Second, when the preset first biomass (od_h) is equal to the second biomass (od_l), the degree of evolution of the cells is reflected by calculating the rate of change of the fed-batch amount.

According to the scheme, the continuous evolution device for the laboratory can realize automatic control and continuous operation of laboratory evolution. In order to obtain accurate biomass data, the biomass can be monitored on line in an external circulation mode. Further, the stability of monitoring is increased by the cooperation of the control system. The invention can obtain the biomass, fed-batch quantity and other data of the cells in the continuous evolution process on line, and after the data are processed by the processor according to the set parameters and the evolution method, the automatic operation of the culture device and the fed-batch device is controlled by the controller module, so that the continuous evolution process of a laboratory can be effectively monitored, and the continuous evolution efficiency of the laboratory is further improved. Therefore, the invention can conveniently realize the continuous evolution process in the laboratory and effectively monitor the evolution process, thereby improving the continuous evolution efficiency of the laboratory.

Drawings

FIG. 1 is a schematic diagram of the composition of a laboratory continuous evolution device in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the internal modules of the controller in an embodiment of the invention.

FIG. 3 is a flow chart of a laboratory continuous evolution method of the present invention implemented using the laboratory continuous evolution apparatus of the present invention.

1-Incubator, 2-blake bottle, 3-magnetic stirrer, 4-feed bottle, 5-feed pump, 6-discharge bottle, 7-discharge pump, 8-first detecting unit, 9-circulating pump, 10-second detecting unit, 11-controller, 12-display screen, 13-knob, 14-magnetic rotor, 15-air filter membrane.

Detailed Description

The invention relates to a continuous evolution device in a laboratory, which consists of a cell culture device, a feeding device, a biomass monitoring device, a feeding monitoring device and a controller. The invention also discloses a continuous evolution method in a laboratory, which can effectively monitor the continuous evolution process, thereby improving the continuous evolution efficiency.

The following detailed description of the invention refers to the accompanying drawings. In which the drawings are for illustrative purposes only and are not intended to be construed as limiting the present patent, and in which certain elements of the drawings may be omitted, enlarged or reduced in order to better illustrate embodiments of the present invention, and not to represent actual product dimensions, it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.

In the description of the present invention, it should be understood that, if there is an orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", etc., which are based on the orientation or positional relationship shown in the drawings, except where specifically noted, it is merely for convenience of describing the present invention and simplifying the description, rather than to indicate or imply that the device or component to be referred must have a specific orientation, be constructed and operated in a specific orientation, and thus the terms describing the positional relationship in the drawings are merely for exemplary illustration and are not to be construed as limitations of the present patent, and that the specific meaning of the terms described above should be understood by those skilled in the art according to specific circumstances.

"Laboratory evolution" refers to the observation of the phenomena of biological evolution by setting the expected conditions in a laboratory and simulating the process of biological evolution by the continuous adaptation of organisms to the living environment. It is particularly suitable for use in microbiological studies, such as for studying the evolutionary potential affecting the phenotype, performance and stability of strains and for rapidly obtaining industrially produced strains containing beneficial mutations, for example by implementing the natural selection rules proposed in Darlichia theory on a laboratory bench to produce evolutionary microbial strains having the desired characteristics.

The present invention differs from the "manual" and "batch" laboratory evolution culture of the prior art in that it enables a "continuous" evolution process, so called "laboratory continuous evolution".

The invention relates to a laboratory continuous evolution device, comprising:

a cell culture device for culturing and evolving cells;

A feeding device for feeding and discharging the culture device;

The biomass monitoring device is arranged to extract a part of the culture solution in the cell culture device and to perform online measurement on the part of the culture solution through the first detection unit so as to obtain biomass data in the culture solution;

the feeding monitoring device is arranged to obtain feeding amount data by measuring the feeding amount or the discharging amount on line;

a controller capable of collecting biomass data and feed-through data and controlling the operation of the evolution device.

As shown in FIG. 1, in one embodiment, the cell culture apparatus of the present invention optionally comprises an incubator 1, particularly an incubator capable of controlling the temperature of the culture fluid, a flask 2 for holding the cell culture fluid, and a stirrer 3 for stirring the culture fluid.

Alternatively, the incubator 1 capable of controlling the temperature of the culture medium includes, but is not limited to, a constant temperature incubator, a constant temperature and humidity incubator, a carbon dioxide incubator, and the like.

Optionally, the culture flask 2 for containing the cell culture solution is a cell culture vessel commonly used in laboratories, including but not limited to shake flasks, screw flasks, etc., and the materials include but are not limited to plastics, glass, stainless steel, etc.

Alternatively, the stirrer 3 for stirring the culture solution includes, but is not limited to, a magnetic stirrer, a shaker which can also realize a stirring function, and the like.

Further alternatively, the concentration of oxygen in the culture broth may be varied by methods including, but not limited to, adding an air filter 15 to the flask, and flushing air, oxygen, nitrogen, carbon dioxide gas, etc. into the flask or incubator.

The feeding device of the present invention optionally includes a feeding bottle 4 and a feeding pump 5 for inflow of the culture solution, and a discharging bottle 6 and a discharging pump 7 for outflow of the culture solution.

The feeding bottle 4 and the discharging bottle 6 can be liquid containers commonly used in laboratories, including but not limited to shake flasks, screw flasks, etc., and materials including but not limited to plastics, glass, stainless steel, etc.

The feed pump 5 for controlling the addition of the culture solution and the discharge pump 7 for controlling the outflow of the culture solution may be selected as peristaltic pumps including, but not limited to, a direct current motor peristaltic pump, a stepper motor peristaltic pump, etc. The feed pump 5 and the discharge pump 7 are preferably provided with an external control interface so as to be controlled by the controller 11.

Further, if the culture solution is easily layered, a stirrer may be added to the bottom of the feed bottle 4. Optionally, the feed bottle 4 may be subjected to a temperature-reducing treatment in order to reduce the risk of contamination of the culture medium in the feed bottle during the long-term continuous evolution. The cooling method includes, but is not limited to, placing the feed bottle 4 in an ice bin.

Still alternatively, two or more of the feed bottle 4 and the feed pump 5 may be provided in the feeding device, and by adjusting the rotation speed ratio between the two or more feed pumps 5, a change in the constituent components in the feed liquid (culture liquid), such as a gradient change, such as a gradient increase, may be achieved.

In the invention, the feeding pump 5 and the discharging pump 7 are independent two pumps, and are respectively controlled by a controller, so that the rotation speed (flow rate) and/or the running time of sample feeding/discharging can be respectively set.

When two or more feed pumps exist, the feed pumps are also respectively controlled by the controller, so that the rotating speed (flow speed) and/or the running time of each path of sample injection can be respectively set.

In one embodiment, the rotational 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 practice, the present invention is not limited to this.

The biomass monitoring device of the invention comprises a first detection unit 8, and partial culture solution is measured through the first detection unit, so that biomass data in the culture solution are obtained. "biomass data" in the present invention refers in particular to the number of cells or the weight of cells per volume of liquid. Biomass can be characterized, for example, by OD values, or by microscopic counting. The first detection unit 8 may thus be selected from a cell turbidimeter or a spectrophotometer, and may be selected from other detection devices that reflect biomass, including but not limited to flow cytometry, microscopy, and the like. The first detection unit 8 is preferably provided with an external communication interface to facilitate communication with the controller 11.

The biomass monitoring device may further comprise a circulation pump 9 for circulating the culture medium. The circulation pump 9 for circulation of the culture solution may be selected from peristaltic pumps including, but not limited to, a DC motor peristaltic pump, a stepper motor peristaltic pump, etc. The circulation pump 9 is preferably provided with an external control interface so as to be controlled by the controller 11.

During the cyclic measurement, a part of the culture medium in the culture flask 2 is pumped out by the circulating pump 9, the biomass data is measured by the first detecting unit 8, and then returned to the culture flask 2.

The feeding monitoring device comprises a second detection unit 10, and obtains feeding amount data by carrying out on-line measurement on feeding or discharging amount. "fed-batch data" in the present invention refers to the amount of liquid added by flow, especially the volume or weight of liquid fed by the feeding device into the cell culture device, or the volume or weight of liquid discharged by the feeding device during one feeding cycle. For example, the second detection unit 10 is an electronic balance. The second detection unit 10 is preferably an external communication interface to facilitate communication with the controller 11. The second detection unit 10 may be arranged to measure the weight change of the feed bottle 4 or the weight change of the feed bottle 6. That is, the culture fluid addition amount can be monitored by measuring the weight change of the feed bottle 4 or by monitoring the weight change of the discharge bottle 6.

In particular, in the case where the culture liquid in the feed bottle 4 needs to be stirred or put in an ice box or the like to be weighed inconveniently, the fed-by amount is preferably monitored by a change in the weight of the discharge bottle 6. When the cell culture temperature is high and the culture solution in the culture flask 2 evaporates more, the fed-batch amount is preferably monitored by the weight change of the feed flask 4.

By means of the second detection unit 10, reliable feed data (volume or weight) can be obtained, which can be used for analyzing/evaluating the state of adaptation and/or evolution for the cells.

Liquid piping aspect. The feed pipeline is that a culture solution feed bottle is connected with a feed pipe inserted at the top of the cell culture bottle through a feed pump. And the discharge pipeline is used for connecting the culture solution discharge bottle with a discharge pipe inserted at the top of the cell culture bottle through a discharge pump. The circulating pipeline is a first detecting unit, a circulating pump and a circulating pipeline, wherein the circulating pipeline is connected with a circulating liquid outlet pipe and a circulating liquid inlet pipe of the cell culture bottle.

It should be noted that the rotation direction of the feed pump is set to the direction in which the culture solution flows into the cell culture flask, and the rotation direction of the discharge pump is set to the direction in which the culture solution flows out of the cell culture flask.

The end of the discharging pipe in the cell culture flask is arranged to have a certain height from the flask bottom of the culture flask, namely, the liquid level height with a certain liquid amount in the culture flask is arranged. In this way, after a certain period of time, the liquid level in the bottle is not drawn out once it falls below the end of the discharge pipe. Thereby maintaining a volume of liquid in the flask, while at this point the end of the tapping pipe is just in contact with the liquid level in the cell culture flask. The tail end of the circulating liquid outlet pipe is arranged at a position closer to the bottle bottom than the discharging pipe, namely the circulating liquid outlet pipe is deep below the liquid level in the cell culture bottle.

The controller can collect biomass data and fed-batch data and control the operation of the evolution device. As shown in fig. 2, in one embodiment, the controller 11 includes a data acquisition module for acquiring data, a data storage module for storing data, a processor module for processing data, and a control module for controlling the operation of other device components, such as the switches and/or speeds of the agitator 3, the feed pump 5, the discharge pump 7, and the circulation pump 9. Further, an external communication module for communicating with the outside may be optionally included. It may optionally also comprise several modules for interaction with the operator, for example a display module for displaying data and/or a parameter setting module for parameter setting. A device start-stop module for controlling system start-up and stop, and/or a power module may also optionally be included.

Further, the data acquisition module can comprise a biomass acquisition module for acquiring cell growth data and a weight acquisition module for acquiring culture fluid addition.

Further, the control module may include a stirrer control module for controlling agitation of the culture solution, a circulation pump control module for controlling circulation of the culture solution, a feed pump control module for controlling inflow of the culture solution from the feed bottle 4 to the cell culture bottle 2, and a discharge pump control module for controlling outflow of the culture solution from the cell culture bottle 2 to the discharge bottle 6.

Further alternatively, the controller is connected to an external computer. For example, the collected data may be stored in the controller and then output to an external computer. For example, the controller may accept parameters and designations from external computer inputs. For example, the modification and upgrade of the continuous evolution implementation in the memory module is also performed after the connection to the external computer.

The laboratory continuous evolution device according to the invention is arranged to perform the method of laboratory continuous evolution according to the invention.

The laboratory continuous evolution method of the invention comprises:

First, parameters are set. The preset parameters may include, but are not limited to, one or more of a first biomass (od_h), a second biomass (od_l), an interval time (Pt), a cycle length (Ct), a feed length (INt), a discharge length (OUTt), etc. In general, the first biomass (od_h) is set to be greater than or equal to the second biomass (od_l). Generally, the outfeed period (OUTt) is set to be greater than or equal to the infeed period (INt).

In the second step, after a certain interval time (Pt), the stirrer stops stirring, and the circulation pump 9 starts to circulate for a period of Ct. After a certain cycle period Ct, the circulation pump 9 stops (i.e., the cycle ends). After a certain cycle time period Ct, the biomass in the portion of the culture fluid in the cycle monitoring system may be considered to be consistent with the biomass of the culture fluid in the cell culture apparatus. After the cycle is completed, the biomass data in the culture broth is measured on line using the first detection unit 8 and the time of data acquisition is recorded.

Third, if the biomass value detected by the first detection unit 8 is smaller than the first biomass (od_h), the stirrer restarts stirring, evolution proceeds, and the second step is continued. If the biomass value detected by the first detection unit 8 is not smaller than the first biomass (od_h), the fourth step is performed.

Fourth, one cycle of feed. The feed pump 5 is started to feed, and the feed pump 5 is operated for a feed duration (INt). Alternatively, the start of the discharge may be started at the same time as the start of the feed, or may be started after waiting a waiting time (Wt) after the start of the feed. The discharge pump 7 is started by starting the discharge pump 7, and the duration of operation of the discharge pump 7 is the discharge duration (OUTt). When the feed pump 5 and the discharge pump 7 are both finished, the fed-batch data are measured on line by the second detection unit 10, and the time of data acquisition is recorded. Then the circulating pump 9 starts to work, the circulating time length is Ct, after the circulation is finished, the first detecting unit 8 is used for measuring the biomass data in the culture solution on line, the time for data acquisition is recorded, and the fifth step is started.

It should be noted that in the present invention, the volume of culture medium in the cell culture flask is determined by the height of the end of the discharge tube from the bottom of the flask. Generally, the workload of the discharge pump is set to be greater than or equal to that of the feed pump, so that the culture solution is pumped to be below the tail end of the discharge pipe every time, and the volume of the culture solution in the culture bottle is kept relatively fixed. The phenomenon that the liquid in the culture bottle is continuously accumulated and increased can not occur. The specific operation can be realized by adjusting the rotation speeds and the working time durations (INt and OUTt) of the feeding pump and the discharging pump. For example, when the pipe diameters and the rotation speeds of the feeding peristaltic pump and the discharging peristaltic pump are the same, the discharging duration OUTt is set to be greater than or equal to the feeding duration INt.

Fifth, if the biomass value detected by the first detecting unit 8 is not smaller than the second biomass (od_h), the fourth step is repeatedly performed, and feeding is continued for a new cycle (new round). If the biomass value detected by the first detection unit 8 is smaller than the second biomass (od_h), the feeding is suspended, the stirrer restarts stirring, evolution is continued, and the second step is performed.

And sixthly, analyzing the collected biomass data and fed-batch data in the evolution process, and evaluating the adaptability and/or the evolution state of the cells.

Specifically, the analysis/evaluation method of the sixth step may have two kinds of:

First, when a preset first biomass (od_h) is greater than a second biomass (od_l), the degree of evolution of the cells is reflected by comparing the change in growth time (Gt) required each time to grow from the second biomass (od_l) to the first biomass (od_h). In general, the shorter the growth time (Gt) required, the better the adaptability of the cells to the culture conditions, the more fully evolved. Meanwhile, the evolution degree of the cells can be further reflected by calculating the rate of change of the fed-batch quantity. The term "rate of change of the fed-batch amount" means a ratio of the amount of change of the total amount of fed/discharged material accumulated after one or more feeding cycles to the time used. For example, at the beginning of evolution, after a number of (e.g., 5) feed cycles, the cumulative feed amount was changed from 50 g to 100 g for 6 hours, i.e., the "rate of change of the fed-batch amount" was 50 g/6 hours. As evolution progressed, the cumulative feed amount was changed from 200 g to 250 g over several (a certain number such as 5) feed periods, and the time taken was 4 hours, i.e. "rate of change of feed amount" was 50 g/4 hours. It is then shown that the adaptation of the cells to the culture conditions is increasingly better. In general, the rate of change of the fed-batch amount is larger and larger, which indicates that the adaptability of the cells to the culture conditions is better and the evolution is more complete.

Second, when the preset first biomass (od_h) is equal to the second biomass (od_l), the degree of evolution of the cells is reflected by calculating the rate of change of the fed-batch amount. The term "rate of change of the fed-batch amount" means a ratio of the amount of change of the total amount of fed/discharged material accumulated after one or more feeding cycles to the time used. For example, at the beginning of evolution, after a number of (e.g., 5) feed cycles, the cumulative feed amount was changed from 50 g to 100 g for 6 hours, i.e., the "rate of change of the fed-batch amount" was 50 g/6 hours. As evolution progressed, the cumulative feed amount was changed from 200 g to 250 g over several (a certain number such as 5) feed periods, and the time taken was 4 hours, i.e. "rate of change of feed amount" was 50 g/4 hours. It is then shown that the adaptation of the cells to the culture conditions is increasingly better. In general, the rate of change of the fed-batch amount is larger and larger, which indicates that the adaptability of the cells to the culture conditions is better and the evolution is more complete.

Optionally, the laboratory continuous evolution method of the present invention may further comprise:

And seventh, based on the result of the analysis/evaluation in the sixth step, changing preset parameters and/or evolution conditions, and then performing the second step.

For example, the modified preset parameters may include, but are not limited to, one or more of a first biomass (OD_H), a second biomass (OD_L), an interval (Pt), a feed duration (INt), a discharge duration (OUTt), a rotational speed (flow rate) of the feed pump, a rotational speed (flow rate) of the discharge pump, and the like.

For example, the modified evolution conditions may be modifying the composition and/or ratio of the feed solution, and/or modifying the culture environment within the incubator, etc.

For example, in a device having two/more feed pumps 5, the ratio between the individual components of the feed solution may be modified by adjusting the rotational speed ratio of the two/more feed pumps 5.

As biological evolution (e.g., microbial evolution) proceeds, the state of cell growth may be too good, resulting in too much total amount of feed-through needed to drop each time from a first biomass (od_h) to a second biomass (od_l), or too short growth time (Gt) needed to grow each time from a second biomass (od_l) to a first biomass (od_h), at which time some parameters or experimental conditions (such as feed solution and/or culture environment) need to be adjusted to better promote/monitor laboratory evolution.

In the other extreme case, the state of cell growth is too poor, resulting in a reduced amount of feed per time from the first biomass (od_h) to the second biomass (od_l) or a too long growth time (Gt) per time from the second biomass (od_l) to the first biomass (od_h), at which time some parameters or experimental conditions (such as feed solution and/or culture environment) need to be adjusted to better promote/detect laboratory evolution.

By means of the method of the invention, a person skilled in the art can conveniently realize controllable and detectable continuous evolution in a laboratory, thereby directionally controlling the evolution direction of microorganisms and obtaining target microbial strains, for example, production strains meeting specific requirements (such as industrial use).

Specific example 1:

The embodiment provides a continuous evolution device in a laboratory. Which connects the hardware devices in the continuous evolution device in the laboratory according to the connection mode in fig. 1.

The cell culture bottle, the magnetic stirrer, the circulating peristaltic pump and the cell turbidimeter are arranged in the constant temperature incubator, and external control interfaces of the magnetic stirrer and the circulating peristaltic pump are respectively connected to a stirrer control module and a circulating pump control module of the controller, and a data interface of the cell turbidimeter is connected to a biomass acquisition module of the controller. In addition, the feeding bottle, the discharging bottle, the feeding peristaltic pump, the discharging peristaltic pump and an electronic balance for obtaining the culture fluid feeding quantity are arranged outside the constant-temperature incubator, external control interfaces of the feeding peristaltic pump and the discharging peristaltic pump are respectively connected to a feeding pump control module and a discharging pump control module of the controller, and a data interface of the electronic balance is connected to a feeding quantity acquisition module of the controller.

It should be noted that, when the volume of the incubator is relatively small, the circulating peristaltic pump and the cell turbidimeter may also be disposed outside the incubator.

Meanwhile, in order to increase the oxygen content in the culture flask during the cell growth process, a pipeline with an air filtering device is arranged at the top of the culture flask in the embodiment.

Taking an evolution experiment of the resistance of escherichia coli to ampicillin as an example, the content of a culture solution in a culture flask is 50 milliliters, the culture solution is LB liquid medium containing ampicillin (10 grams of peptone, 5 grams of yeast powder, 10 grams of sodium chloride per liter of LB liquid medium, the pH is adjusted to 7.2 by sodium hydroxide or dilute hydrochloric acid, and the culture flask is autoclaved for 30 minutes), and the initial concentration of the ampicillin is 1 microgram/milliliter.

In the embodiment, the silica gel tube used in all the liquid pipelines has the specification of 2mm inside diameter and 4mm outside diameter, the rotating speed of the magnetic stirrer is set to 200 rpm, and the rotating speed of the circulating peristaltic pump is set to 100 ml/min. The culture medium in one feed bottle was LB liquid medium containing 1. Mu.g/ml ampicillin, and the corresponding initial rotational speed of the feed peristaltic pump was set at 50 ml/min. The rotational speed of the discharge peristaltic pump was set at 50 ml/min.

Optionally, a second feeding bottle is also arranged, the culture solution in the second feeding bottle is LB liquid medium containing 100 micrograms/milliliter of ampicillin, and the initial rotating speed of the corresponding feeding peristaltic pump is set to be 0 milliliter/minute.

In this embodiment, the modules inside the controller are connected according to the connection mode shown in fig. 2. The parameter setting module is connected with the data storage module, and the set parameters are stored in the storage module. The parameter setting is performed by means of a knob mounted on the controller. The storage module is connected with the data acquisition module with the RS 232-to-TTL interface to acquire biomass data and fed-batch data. The storage module simultaneously stores preset continuous evolution implementation modes. The processor module obtains the data in the data storage module and processes the data according to the stored implementation and parameters. According to the result of data processing, the processor respectively controls the running states of the magnetic stirrer, the circulating peristaltic pump, the feeding peristaltic pump and the discharging peristaltic pump by changing the state of the electromagnetic relay in the control module. The operation parameters and the data collected in the operation process can be displayed through the LCD display module.

It should be noted that the storage module may be connected to an external computer through a USB interface on the external communication module. At this time, the setting of the parameters can be performed by an external computer, and the collected data can also be output to the external computer by an external communication module. In addition, the modification and upgrade of the continuous evolution implementation in the memory module also needs to be performed after the connection with an external computer. The equipment start-stop module is connected with the processor module to control the start and stop of the whole system.

Specific example 2:

The present invention provides a method for continuous evolution in a laboratory, which is described below with reference to fig. 3.

FIG. 3 is a flow chart of a laboratory continuous evolution method according to an embodiment of the present invention.

Also taking the evolution experiment of the escherichia coli on ampicillin resistance as an example, the second biomass (od_l) was set to 0.5, the first biomass (od_h) was set to 1.5, the interval time (Pt) was 10 minutes, the cycle time (Ct) was 15 seconds, the feeding time (INt) was 10 seconds, and the discharging time (OUTt) was 20 seconds.

After the completion of the parameter setting, E.coli cultured overnight was inoculated into the flask in an inoculum size of 0.1OD. The start button on the controller panel is pressed, at which time the system starts to run and the agitator starts to operate. After the system is operated for 10 minutes, the stirrer stops stirring, the circulating pump starts to circulate, and the circulation time is 15 seconds. After the circulation is finished, biomass in the culture solution is measured on line through a cell turbidimeter, data are sent to a biomass acquisition module of the controller, and the acquisition module simultaneously sends the numerical value of the biomass and the acquisition time to a data storage module.

The processor obtains the data in the data storage module and compares the relationship between the current measured biomass value and the set first biomass (od_h). When the biomass value in the online measured culture solution is less than 1.5, the stirrer restarts stirring, and the cell culture is continued. The above operation was repeated after 10 minutes of system operation. When the biomass number in the on-line measured broth is greater than 1.5, it is indicated that the system has completed a round of cultivation. At this time, the feed pump starts feeding, the feeding duration is 10 seconds, the discharge pump starts discharging, and the discharging duration is 20 seconds. And the electronic balance is used for measuring the fed-batch quantity of the culture solution on line and sending the data to a fed-batch quantity acquisition module of the controller, and the acquisition module is used for simultaneously sending the fed-batch quantity value and the acquisition time to the data storage module. And then the circulating pump starts to work, the circulating time is 15 seconds, after the circulation is finished, the cell turbidimeter measures the biomass in the culture solution on line, and sends the data to a biomass acquisition module of the controller, and the acquisition module simultaneously sends the value of the biomass and the acquisition time to a data storage module. 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 online measured culture solution is greater than 0.5, the above process is repeated until the biomass value in the online measured culture solution is less than 0.5 of the second biomass. At this point the system will initiate a new round of evolution work.

As evolution proceeds, the time required for each cell to grow from the second biomass (od_l) to the first biomass (od_h) becomes progressively shorter, with an increasing rate of change in the feed. At this time, the ampicillin concentration in the feed medium was adjusted and the evolution was continued. The evolution is continued, for example, by manually changing the feed broth or by adjusting the speed ratio of the two feed peristaltic pumps to increase the ampicillin concentration in the mixed feed broth to 10. Mu.g/ml.

It is worth mentioning that, as the concentration of the antibiotics in the culture solution is continuously increased, when the situation that the cells cannot grow for a long time (such as the biomass data and the fed-batch data monitored on line are unchanged for more than 24 hours) appears, the increasing amplitude of the antibiotics is too large, and at this time, the concentration of the antibiotics in the mixed feed culture solution can be reduced to enable the cells to recover to grow and continue to evolve. The ampicillin concentration in the feed broth was reduced, for example, by manually changing the feed broth, or by adjusting the ratio of the rotational speeds of the two feed peristaltic pumps.

The above embodiments are only for illustrating the technical solution of the present invention, not for limiting the same, and 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 modifications may be made to the technical solution described in the above embodiments or equivalents may be substituted for some of the technical features thereof, and those modifications or substitutions,

The essence of the corresponding technical scheme is not deviated from the protection scope of the technical scheme of each embodiment of the invention.

Claims (10)

1. A laboratory continuous evolution device comprising:

a cell culture device for culturing and evolving cells;

A feeding device for feeding and discharging the culture device;

The biomass monitoring device is arranged to extract a part of the culture solution in the cell culture device and to perform online measurement on the part of the culture solution through the first detection unit so as to obtain biomass data in the culture solution;

the feeding monitoring device is arranged to obtain feeding amount data by measuring the feeding amount or the discharging amount on line;

a controller capable of collecting biomass data and feed-through data and controlling the operation of the evolution device.

2. The laboratory evolution device according to claim 1, wherein the biomass data and the fed-batch data it obtains are used for analyzing/assessing the adaptability and/or the state of evolution of cells.

3. The laboratory evolution device of claim 1, wherein the second detection unit is an electronic balance for measuring weight changes of the feed bottle or the discharge bottle.

4. The laboratory evolution device according to claim 1, wherein the feeding monitoring device comprises a feeding pipeline and a discharging pipeline, the feeding pipeline comprises a feeding pump and a feeding pipe, the discharging pipeline comprises a discharging pipe and a discharging pump, one end of the discharging pipe is positioned in the culture bottle, the tail end of the discharging pipe positioned in the culture bottle is arranged to be at a certain height from the bottle bottom of the culture bottle, the feeding pump and the discharging pump are respectively controlled by the controller, and the workload of the discharging pump is larger than or equal to that of the feeding pump in one feeding period.

5. The laboratory evolution device according to claim 1, wherein the feed-in monitoring device has two/more feed pumps, the flow rate and/or the run time of each feed can be set separately.

6. A laboratory continuous evolution method using the laboratory continuous evolution apparatus according to any one of claims 1 to 5, the method comprising:

Firstly, setting parameters;

The second step, after a certain interval time, the stirrer stops stirring, the circulating pump starts circulating, after the circulation is finished, the first detection unit is used for measuring biomass data in the culture solution, and the time for data acquisition is recorded;

a third step of restarting stirring by the stirrer if the biomass value detected by the first detection unit is smaller than the first biomass (OD_H), continuing evolution, and continuing to execute the second step;

Starting a feeding pump to feed for a certain feeding time period, starting a discharging pump to discharge at the same time or later, and continuously discharging for a certain discharging time period, after the feeding pump and the discharging pump are finished working, measuring fed-batch data on line by a second detection unit, and recording the time of data acquisition;

a fifth step of repeating the fourth step to continue feeding for a new period if the biomass value detected by the first detecting unit is not less than the second biomass (OD_L), and suspending feeding if the biomass value detected by the first detecting unit is less than the second biomass, restarting stirring by the stirrer to continue evolution, and executing the second step;

and sixthly, analyzing the collected biomass data and fed-batch data in the evolution process, and evaluating the adaptability and/or the evolution state of the cells.

7. The laboratory continuous evolution method of claim 6, further comprising

And seventh, based on the result of the analysis/evaluation in the sixth step, changing preset parameters and/or evolution conditions, and then performing the second step.

8. The laboratory continuous evolution method of claim 7, wherein altering the evolution conditions can be altering the composition and/or the ratio of the feed solution, and/or altering the culture environment within the incubator.

9. The laboratory continuous evolution method of claim 8, wherein altering the evolution conditions is altering the feed solution ratio by adjusting the speed ratio of the two/more feed pumps, thereby altering the ratio between the individual components of the feed solution.

10. The laboratory continuous evolution method of claim 6, wherein the analysis/evaluation method of the sixth step comprises one of:

First, when the preset first biomass (OD_H) is larger than the second biomass (OD_L), the evolution degree of the cells is reflected by comparing the change of the growth time (Gt) required for each growth from the second biomass (OD_L) to the first biomass (OD_H);

Second, when the preset first biomass (od_h) is equal to the second biomass (od_l), the degree of evolution of the cells is reflected by calculating the rate of change of the fed-batch amount.