Control method for realizing in-vitro biosynthesis

Abstract

The invention discloses a control method for realizing in-vitro biosynthesis, which comprises the following steps: after starting, a detection part continuously detects to obtain detection parameters; feeding according to the stored preset feeding amount, and judging whether a feeding completion signal is generated or not according to a preset feeding condition after feeding is completed; when a preset feeding condition is met, generating a feeding completion signal and carrying out a reaction; in the reaction process, according to the detection parameters, continuously carrying out condition judgment on whether a preset monitoring condition is met or not; when the preset monitoring condition is not met, selecting a corresponding preset action; after the reaction is completed, generating a reaction completion signal and stopping the reaction; and material taking is controlled according to the reaction completion signal, and a material taking completion signal is generated after material taking is completed. According to the in-vitro biosynthesis control system, the whole process of in-vitro biosynthesis can be automatically controlled, meanwhile, the reaction quality is automatically controlled, the reaction process and the reaction quality are monitored at any time by presetting related parameters, and corresponding preset actions can be provided for the conditions which do not meet the conditions.

Description

Technical Field

[0001] The present invention relates to the field of in vitro biosynthesis, and particularly relates to a control method for realizing in vitro biosynthesis. Background Art

[0002] In vitro biosynthesis, such as cell-free in vitro biosynthesis - obtaining the basic components required for transcription and translation in cells, adding a DNA template in vitro to maintain the operation of gene transcription, protein translation or metabolic processes, so as to synthesize a target product. Cell-free synthetic biology (CFSE) removes the cell membrane and can directly regulate the internal life activities of cells; removes the natural genome, eliminates the regulation of non-essential genes, and decouples cell growth and core metabolic regulation; the system is open, there is no material transport obstacle, it is easy to add substrates, remove products and monitor and analyze the process, and has the greatest degree of freedom in engineering, playing an important role in both basic disciplines and engineering applications: revealing life systems such as protein translation mechanisms; having broad application potential in the fields of structural biology, high-throughput screening, biocatalysis, biomedicine, etc.

[0003] Currently, most in vitro biosynthesis processes use open stirred tanks, which rely on ventilation pipes to blow cold and hot air to control the temperature of the reaction solution, disperse the gas generated by the reaction solution to reduce the interference of the gas on the reaction, and extract small samples of the reaction solution for manual detection and other measures to control the quality of the reaction solution. The operation is not convenient and time-consuming, and the quality control of the reaction solution cannot be achieved in real time. Operators need to pay attention to the situation of the reaction solution at any time and make corresponding operations. Moreover, there are a wide variety of target products in in vitro biosynthesis, and production operators need to adjust intervention conditions according to the types of target products. Restricted by human factors, it is extremely easy to make the overall quality control of the reaction solution unstable, and there is a risk of contact exposure in the open manual operation process, and at the same time, it also increases the possibility of reaction solution contamination, causing unnecessary impacts. Therefore, there is an urgent need for a control method for realizing in vitro biosynthesis to solve the above problems. Summary of the Invention

[0004] To solve the above technical problems, a control method for realizing in vitro biosynthesis provided by the present invention includes: after startup, a detection unit continuously detects detection parameters; according to a preset feed amount stored, a feed pump is controlled to pump reaction raw materials into a tank for reaction through a feed pipe, and after the feeding is completed, it is judged whether a feeding completion signal is generated according to preset feeding conditions; when the preset feeding conditions are met, the feeding completion signal is generated, and then the tank is driven to rotate for reaction according to a preset reaction time and a preset reaction speed; during the reaction process, it is continuously judged whether the conditions meet preset monitoring conditions according to the detection parameters; when the preset monitoring conditions are not met, corresponding preset actions are selected; when the rotation reaches the preset reaction time, the rotation of the tank is controlled to stop, and a reaction completion signal is generated; according to the reaction completion signal, a material taking pump is controlled to discharge the reaction product out of the tank through a material taking pipe, and a material taking completion signal is generated after the material taking is completed.

[0005] Specifically, the detection parameters are detection parameters obtained by detecting the reaction solution and / or the gas generated during the reaction, and the detection parameters are any one or more of the following: a reaction solution temperature parameter obtained by detecting the temperature of the reaction solution, an ethanol gas concentration parameter obtained by detecting the ethanol concentration of the gas generated in the reaction, and a reaction solution activity parameter obtained by detecting the activity of the reaction solution.

[0006] Specifically, the reaction solution activity parameter is any one or more of a pH parameter obtained by detecting the pH of the reaction solution through a pH sensor, a dissolved oxygen parameter obtained by detecting the dissolved oxygen of the reaction solution through a fluorescence dissolved oxygen sensor, a turbidity parameter obtained by detecting the turbidity of the reaction solution through a turbidity sensor, and a conductivity parameter obtained by detecting the conductivity of the reaction solution through a conductivity sensor.

[0007] Specifically, the situations where the preset monitoring conditions are not met include any one or more of the following:

[0008] The first one: the duration for which at least one of the reaction solution activity parameters does not meet the preset activity range exceeds the preset failure time range. At this time, the corresponding preset actions include generating a reaction failure signal and controlling the tank to stop rotating;

[0009] The second one: the reaction solution temperature parameter does not meet the preset temperature range. At this time, the corresponding preset actions include starting a circulation temperature control component to perform temperature control adjustment of the reaction solution, and judging whether the duration for which the reaction solution temperature parameter does not meet the preset temperature range meets the automatic recovery time range for the preset temperature non - compliance. If not, the rotation of the tank is controlled to stop; if so, the rotation of the tank is controlled to continue;

[0010] The third case: The duration for which the temperature parameter of the reaction solution does not conform to the preset temperature range exceeds the stop time range for non - conformity of the preset temperature. At this time, the corresponding preset action includes determining whether the activity parameter of the reaction solution conforms to the preset activity range. If it does not conform, the reaction failure signal is generated and the rotation of the tank body is controlled to stop. If it conforms, the rotation of the tank body is controlled to continue;

[0011] The fourth case: The ethanol gas concentration parameter does not conform to the preset ethanol concentration range. At this time, the corresponding preset action is to control the gas regulation component to adjust the gas concentration, and determine whether the duration for which the ethanol gas concentration parameter does not conform to the preset ethanol concentration range conforms to the automatic recovery time range for non - conformity of the preset ethanol concentration. If it does not conform, the rotation of the tank body is controlled to stop. If it conforms, the rotation of the tank body is controlled to continue;

[0012] The fifth case: The duration for which the ethanol gas concentration parameter does not conform to the preset ethanol concentration range exceeds the stop time range for non - conformity of the preset ethanol concentration. At this time, the corresponding preset action includes determining whether the activity parameter of the reaction solution conforms to the preset activity range. If it does not conform, the reaction failure signal is generated and the rotation of the tank body is controlled to stop. If it conforms, the rotation of the tank body is controlled to continue.

[0013] Specifically, the preset feeding condition is that the temperature parameter of the reaction solution conforms to the preset reaction solution temperature range and / or the ethanol gas concentration parameter conforms to the preset ethanol gas concentration range; Preferably, when the temperature parameter of the reaction solution does not conform to the preset reaction solution temperature range, the tank body starts the circulating temperature control component to perform temperature control adjustment of the reaction solution; when the ethanol gas concentration parameter does not conform to the preset ethanol gas concentration range, the tank body controls the gas regulation component to adjust the gas concentration.

[0014] Specifically, according to the failed reaction signal, the material taking pump is controlled to discharge the reaction product from the tank body through the material taking pipe, and the material taking completion signal is generated after the material taking is completed.

[0015] Specifically, when the material taking completion signal is generated, the cleaning component is controlled to perform cleaning according to the stored automatic cleaning parameters.

[0016] Specifically, the automatic cleaning parameters include: the individual cleaning feeding operation time, the cleaning feeding reaction material taking common operation time, or the individual cleaning material taking operation time for a preset number of seconds.

[0017] Specifically, the preset feed rate, the preset reaction time, the preset reaction rate, the preset reaction liquid temperature range, the preset ethanol gas concentration range, the automatic recovery time range for non-compliance with the preset temperature, the stop time range for non-compliance with the preset temperature, the automatic recovery time range for non-compliance with the preset ethanol concentration, and the stop time range for non-compliance with the preset ethanol concentration are stored as an operation data packet corresponding to different target products.

[0018] Specifically, the operation data packet has two modes: pre-stored corresponding to the production model or input and stored during use corresponding to custom production. The production model refers to the operation data packet with fixed values under different production processes corresponding to different types of target products. If various parameters under the production model need to be modified, it is necessary to switch to the custom production mode for modification.

[0019] Specifically, it also includes terminating the reaction with one key through an emergency stop button.

[0020] Specifically, when controlling the feed pump to pump the reaction raw materials into the tank for reaction through the feed pipe, the current device weight is read from the weighing sensor of the tank and compared with the weight before feeding, and the remaining feed amount is queried in real time and the feeding progress is displayed; when controlling the take-out pump to discharge the reaction product from the tank through the take-out pipe, the current device weight is read from the weighing sensor of the tank and compared with the weight before taking out, and the remaining take-out amount is queried in real time and the take-out progress is displayed.

[0021] The present invention also provides an in vitro biosynthesis system for implementing the control method of in vitro biosynthesis in the foregoing technical solution.

[0022] Specifically, it includes: a tank body assembly, a rotating assembly, a feeding assembly, a reaction raw material quantity feedback unit, a detection unit, and a control device communicatively connected to the foregoing components. Among them, the tank body assembly has a tank body for accommodating a reaction solution for in vitro biological reactions; the rotating assembly is used to achieve the mixing of the reaction solution; the feeding assembly has a feeding pipe connected to the tank body and a feeding pump connected to the feeding pipe; the reaction raw material quantity feedback unit is used to detect the raw material quantity in the tank body to obtain a raw material quantity parameter; the detection unit is used to detect the reaction solution and / or the gas generated by the reaction to obtain a detection parameter; the control device has a feeding module, an operation module, and a mixing module; wherein, the feeding module controls the feeding pump to pump the reaction raw material into the tank body through the feeding pipe; the operation module obtains the raw material quantity parameter and determines whether it meets the start condition at least according to the raw material quantity parameter. If so, it generates a start signal and sends it to the mixing module; after receiving the start signal, the rotating module starts the rotating assembly to rotate for a predetermined time and at a predetermined speed; the operation module is also communicatively connected to the detection unit to receive the detection parameter and controls the reaction process according to the detection parameter.

[0023] Advantages of the present invention:

[0024] 1. The control method provided by the present invention can realize the automatic control of in vitro biosynthesis, realize the whole process automation of automatic feeding, automatic reaction, automatic detection, and automatic material taking, and at the same time realize the automatic control of the reaction quality. By presetting relevant parameters, the reaction process and the quality of the reaction are monitored at all times, and corresponding preset actions can be provided for situations that do not meet the conditions.

[0025] 2. The control method provided by the present invention can realize a foolproof operation, further reducing the workload and operation knowledge requirements of operators.

[0026] 3. When the control method provided by the present invention is applied to actual production, it can first conduct a trial production, then save the parameters, and perform a one-key production model production, meeting the free switching from sample trial production to mass production of products. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for the present invention are briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a schematic diagram of the control method of the present invention.

[0029] Figure 2It is the judgment process of whether the activity parameters of the reaction solution of the present invention meet the preset monitoring conditions.

[0030] Figure 3 It is the judgment process of whether the temperature parameter of the reaction solution and the ethanol gas concentration parameter of the present invention meet the preset monitoring conditions.

[0031] Figure 4 It is the overall assembly and disassembly diagram of the in vitro biosynthesis reaction system.

[0032] Figure 5 It is the schematic diagram of the assembly and disassembly of the reaction tank assembly and part of the circulation temperature control assembly.

[0033] Figure 6 It is the schematic diagram of the coil pipe.

[0034] Figure 7 It is the schematic diagram of the assembly and disassembly of the circulation temperature control assembly.

[0035] Figure 8 It is another schematic diagram of the circulation temperature control assembly.

[0036] Figure 9 It is the cross-sectional view of the connection relationship between the liquid detection component and the reaction tank component.

[0037] Figure 10 It is the cross-sectional view of the large ventilation pipe.

[0038] Figure 11 It is the assembly and disassembly diagram of the ethanol gas concentration detection device.

[0039] Figure 12 It is the structural schematic diagram of the on-line quantitative detection equipment for reaction products.

[0040] Figure 13 It is Figure 12 The cross-sectional view of the light source irradiation channel in

[0041] Among them, the reference numerals in the figures are as follows: 1. outer shell assembly, 2. structural support frame, 3. reaction tank assembly, 31. tank body, 32. transparent monitoring door, 33. end cover, 34. roller track, 35. stirring blade, 36. tail seal plate, 361. slip ring connector, 37. gear connection plate, 38. large gear, 39. slip ring, 4. gas detection assembly, 41. large ventilation pipe, 42. sealing bearing, 43. fixed seat of large ventilation pipe, 44. extraction pipe, 45. feed connection ferrule, 46. first extraction pipe, 47. transfer detection chamber, 471. ethanol sensor, 472. sealing gasket, 48. second extraction pipe, 49. extraction component, 5. liquid detection assembly, 51. detection box body, 52. sensor, 53. float switch, 54. liquid detection pipeline, 6. circulation temperature control assembly, 61. coil pipe, 62. coil pipe pressing strip, 63. circulation temperature control pipeline, 64. constant temperature medium liquid tank, 65. sponge, 66. medium circulation pump, 7. on-line quantitative detection equipment for reaction products, 71. detection device, 72. optical device, 73. optical path setting, 74. detection chamber, 75. light source, 76. sample flow pipeline, 77. liquid inlet section and liquid outlet section, 78. sample detection pipe, 8. feed pipe. Detailed implementation manners

[0042] Next, the technical solutions in the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] As Figure 1 shown, the present invention provides a control method for realizing in vitro biosynthesis, including: after startup, the detection unit continuously detects to obtain detection parameters; according to the stored preset feed amount, controls the feed pump to pump the reaction raw materials into the tank body for reaction through the feed pipe, and judges whether a feed completion signal is generated according to the preset feed conditions after the feed is completed; when meeting the preset feed conditions, generates the feed completion signal, and then drives the tank body to rotate for reaction according to the preset reaction time and preset reaction speed; during the reaction process, continuously judges whether it meets the preset monitoring conditions according to the detection parameters; when not meeting the preset monitoring conditions, selects corresponding preset actions; when the rotation reaches the preset reaction time, controls the tank body to stop rotating and generates a reaction completion signal; according to the reaction completion signal, controls the material taking pump to discharge the reaction products from the tank body through the material taking pipe, and generates a material taking completion signal after the material taking is completed.

[0044] In one embodiment, the detection parameter is a detection parameter obtained by detecting the reaction solution and / or the gas generated during the reaction. The detection parameter is any one or more of the following: the reaction solution temperature parameter obtained by detecting the reaction solution temperature, the ethanol gas concentration parameter obtained by detecting the ethanol concentration of the gas generated during the reaction, and the reaction solution activity parameter obtained by detecting the activity of the reaction solution. Specifically, the acquisition of the detection parameter can be continuous or intermittent. The reaction process of the present invention further includes the feeding of the reaction. It should be noted that the reaction solution temperature parameter and the ethanol gas concentration parameter need to be detected not only during the reaction but also during the preparation process of the reaction, such as feeding.

[0045] In one embodiment, the reaction solution activity parameter is any one or more of the pH parameter obtained by detecting the pH of the reaction solution through a pH sensor, the dissolved oxygen parameter obtained by detecting the dissolved oxygen of the reaction solution through a fluorescence dissolved oxygen sensor, the turbidity parameter obtained by detecting the turbidity of the reaction solution through a turbidity sensor, and the conductivity parameter obtained by detecting the conductivity of the reaction solution through a conductivity sensor. Specifically, the reaction solution activity parameter is a combination of the actual detection values of various sensors corresponding to a preset activity range. The preset activity range can be set according to the properties of the reaction solution. Whether the reaction solution activity parameter conforms to the preset activity range is the key to controlling the quality of in vitro biosynthesis.

[0046] In one embodiment, the situations that do not meet the preset monitoring conditions are any one or more of the following:

[0047] The first type: The duration for which at least one reaction solution activity parameter does not conform to the preset activity range exceeds the preset failure time range. In this case, the corresponding preset action includes generating a reaction failure signal and controlling the tank to stop rotating;

[0048] The second type: The reaction solution temperature parameter does not conform to the preset temperature range. In this case, the corresponding preset action includes starting the circulation temperature control component to adjust the temperature control of the reaction solution, and determining whether the duration for which the reaction solution temperature parameter does not conform to the preset temperature range meets the automatic recovery time range for the preset temperature non - compliance. If not, control the tank to stop rotating; if so, control the tank to continue rotating;

[0049] The third type: The duration for which the reaction solution temperature parameter does not conform to the preset temperature range exceeds the preset stop time range for the preset temperature non - compliance. In this case, the corresponding preset action includes determining whether the reaction solution activity parameter conforms to the preset activity range. If not, generate a reaction failure signal and control the tank to stop rotating; if so, control the tank to continue rotating;

[0050] The fourth case: The ethanol gas concentration parameter does not fall within the preset ethanol concentration range. In this case, the corresponding preset action is to control the gas regulation component to adjust the gas concentration, and determine whether the duration for which the ethanol gas concentration parameter does not fall within the preset ethanol concentration range meets the automatic recovery time range for the preset non-compliance of ethanol concentration. If not, control the tank body to stop rotating; if so, control the tank body to continue rotating.

[0051] The fifth case: The duration for which the ethanol gas concentration parameter does not fall within the preset ethanol concentration range exceeds the stop time range for the preset non-compliance of ethanol concentration. In this case, the corresponding preset actions include determining whether the reaction liquid activity parameter meets the preset activity range. If not, generate a reaction failure signal and control the tank body to stop rotating; if so, control the tank body to continue rotating.

[0052] Specifically, as Figures 2 - 3 shown, the conditional judgment of the detection parameters includes: whether the reaction liquid activity parameter meets the preset monitoring condition and whether the reaction liquid temperature parameter and ethanol gas concentration parameter meet the preset monitoring condition.

[0053] When judging the activity of the reaction liquid, when the reaction liquid activity parameter does not exceed the preset activity range, the reaction liquid activity is valid and the reaction continues; when the physical and chemical parameters of the reaction liquid exceed the preset activity range, the reaction liquid activity fails; further, when the duration for which the reaction liquid activity fails does not exceed the preset failure time range, the reaction has not failed and the reaction continues; if it exceeds, the reaction fails.

[0054] For the judgment of reaction activity, at least one physical and chemical parameter can be selected according to different target products, that is, a single physical and chemical parameter can be selected or multiple physical and chemical parameters can be selected. Each physical and chemical parameter has a preset activity range corresponding to different target products. Therefore, the activity being valid means that each reaction liquid physical and chemical parameter index corresponding to the preset activity range needs to be within its corresponding preset activity range. For example, the preset activity ranges are pH value A, conductivity value B, fluorescence dissolved oxygen value C, and turbidity value D, corresponding to 4 different physical and chemical parameters respectively. Only when each reaction liquid physical and chemical parameter corresponding to the preset activity range meets its respective preset activity range is the reaction liquid activity valid; otherwise, as long as one does not meet the range, it is considered that the reaction activity fails. Further, when judging that the reaction liquid activity fails, if the preset reaction liquid activity failure time range is not more than 500 seconds, then when the duration for which the reaction liquid activity fails does not exceed 500 seconds, the operation module controls the reaction to continue; when the duration for which the reaction liquid activity fails exceeds 500 seconds, the operation module determines that the reaction fails and controls the reaction to terminate.

[0055] When judging the reaction liquid temperature parameter and / or the ethanol gas concentration parameter, when the reaction liquid temperature parameter and / or the ethanol gas concentration parameter do not conform to the preset temperature range and / or the preset ethanol concentration range, start to regulate the corresponding temperature control component and / or the gas regulation component; further, when the duration of non-conformance does not exceed the corresponding preset temperature non-conformance automatic recovery time range and / or the preset ethanol concentration non-conformance automatic recovery time range, the reaction continues; when the duration of non-conformance exceeds the corresponding preset temperature automatic recovery time range and / or the preset gas concentration automatic recovery time range, the reaction stops;

[0056] Furthermore, when the duration of non-conformance does not exceed the corresponding preset temperature non-conformance stop time range and / or the preset ethanol concentration non-conformance stop time range, the reaction resumes automatically and continues; when the duration of non-conformance exceeds the corresponding preset temperature non-conformance stop time range and / or the preset ethanol concentration non-conformance stop time range, it is necessary to judge whether the reaction liquid activity parameter conforms to the preset activity range. If it conforms, the reaction continues; if it does not conform, the reaction fails.

[0057] According to the above embodiments, the preset temperature automatic recovery time range is not greater than the preset temperature stop time range; the preset ethanol concentration automatic recovery time range is not greater than the preset ethanol concentration stop time range; the preset temperature automatic recovery time range and the preset ethanol concentration automatic recovery time range can be the same; the preset temperature stop time range and the preset ethanol concentration stop time range can be the same.

[0058] In one embodiment, the preset feeding conditions are that the temperature parameter of the reaction solution conforms to the preset reaction solution temperature range and / or the ethanol gas concentration parameter conforms to the preset ethanol gas concentration range; preferably, when the temperature parameter of the reaction solution does not conform to the preset reaction solution temperature range, the tank starts the circulation temperature control component to adjust the temperature control of the reaction solution; when the ethanol gas concentration parameter does not conform to the preset ethanol gas concentration range, the tank controls the gas regulation component to adjust the gas concentration. Specifically, taking the production of in vitro synthesized protein as an example, the biological reaction solution for in vitro biosynthesis is extremely sensitive to temperature. Usually, the protein raw material is stored in an environment of -18 degrees, and it also needs to be stored at a low temperature after being mixed into the reaction solution. The biological reaction solution is promoted to react by heating, but too high a temperature will cause protein inactivation, and too low a temperature will affect the reaction efficiency, both of which will affect the activity of the reaction solution. And the current manual detection of the reaction solution temperature is restricted by human factors, and the detection result of the reaction solution temperature cannot be updated in real time, which is not conducive to the improvement of the reaction efficiency and the control of the reaction quality. Ethanol gas is released during the reaction of the biological reaction solution. Too high an ethanol gas concentration will inhibit the protein reaction. Therefore, the reaction solution needs to be stirred during the production process to fully react and precipitate ethanol gas. To sum up, ensuring that the ethanol gas concentration of the reaction solution is below the safety threshold and ensuring that the temperature of the reaction solution does not exceed the preset temperature threshold range are the keys to improving the quality of the reaction solution for most biological reaction solution stirring reactions. And this application incorporates the reaction solution temperature and ethanol gas concentration into the feeding conditions, which is conducive to strictly controlling the reaction quality at the initial stage and improving the reaction efficiency.

[0059] In one embodiment, according to the failed reaction signal, the material taking pump is controlled to discharge the reaction product out of the tank through the material taking pipe, and a material taking completion signal is generated after the material taking is completed. Specifically, in addition to the reaction completion signal, the reaction failure signal also controls the material taking pump to discharge the reaction product out of the tank through the material taking pipe, and can timely process the reaction solution with failed reaction.

[0060] In one embodiment, when the material taking completion signal is generated, the cleaning component is controlled to perform cleaning according to the stored automatic cleaning parameters. Specifically, the cleaning component can also input the cleaning liquid into the tank through the feeding pipe by controlling the feeding pump, and discharge the cleaning liquid out of the tank through the material taking pipe by controlling the material taking pump.

[0061] In one embodiment, the automatic cleaning parameters include: the individual cleaning feeding operation time of a preset number of seconds, the common operation time of cleaning feeding reaction and material taking, or the individual cleaning material taking operation time. Specifically, for different cleaning requirements, different cleaning parameters are selected to complete the automatic cleaning process.

[0062] In one embodiment, a preset feed rate, a preset reaction time, a preset reaction rate, a preset reaction liquid temperature range, a preset ethanol gas concentration range, a preset automatic recovery time range for temperature non - compliance, a preset stop time range for temperature non - compliance, a preset automatic recovery time range for ethanol concentration non - compliance, and a preset stop time range for ethanol concentration non - compliance are stored as an operation data packet corresponding to different target products. Specifically, relevant parameters to be controlled are stored in the operation data packet to achieve different condition controls for in vitro biosynthesis reactions.

[0063] In one embodiment, the operation data packet has two modes: pre - stored corresponding to a production model or input - stored during use corresponding to custom production. A production model refers to an operation data packet with fixed values under different production processes corresponding to different types of target products. If various parameters under the production model need to be modified, it is necessary to switch to the custom production mode for modification. Specifically, the control method of the present invention can manage detection parameters according to the operation data packet of the production model, enabling fully automatic quality - controlled production. At the same time, it can test whether the detection parameters are reasonable according to the operation data packet of custom production, adapting to both large - scale production and small - dose trial production. When the control method provided by the present invention is applied to actual production, it can first conduct trial production, then save the parameters, and perform one - key production model production, meeting the free switching from sample trial production to quantitative production of products.

[0064] In one embodiment, it also includes terminating the reaction with one key through an emergency stop button. Specifically, when emergency handling is required, the control method provided by the present invention can also perform emergency operations through the emergency stop button.

[0065] In one embodiment, when controlling the feed pump to pump reaction raw materials into the tank for reaction through the feed pipe, the current weight of the device and the weight before feeding are read from the weighing sensor of the tank for comparison, and the remaining feed amount is queried in real - time and the feeding progress is displayed; when controlling the take - out pump to discharge the reaction product from the tank through the take - out pipe, the current weight of the device and the weight before taking out are read from the weighing sensor of the tank for comparison, and the remaining take - out amount is queried in real - time and the take - out progress is displayed.

[0066] The present invention also provides a system for in vitro biosynthesis, which is used to implement the control method for in vitro biosynthesis in the foregoing technical solution.

[0067] In a specific embodiment, the in vitro biosynthesis system includes: a reaction tank assembly 3, a rotation assembly, a feeding assembly, a reaction raw material quantity feedback unit, a detection unit, and a control device communicatively connected to the foregoing components. Among them, the reaction tank assembly 3 has a tank body 31 for containing a reaction solution to perform an in vitro biological reaction; the reaction rotation assembly is used to achieve the mixing of the reaction solution; the feeding assembly has a feeding pipe 8 connected to the tank body 31 and a feeding pump connected to the feeding pipe 8; the reaction raw material quantity feedback unit is used to detect the quantity of raw materials in the tank body 31 to obtain a raw material quantity parameter; the detection unit is used to detect the reaction solution and / or the gas generated by the reaction to obtain a detection parameter; the control device has a feeding module, an operation module, and a mixing module. Among them, the feeding module controls the feeding pump to pump the reaction raw materials into the tank body through the feeding pipe 8; the operation module obtains the raw material quantity parameter and determines whether it meets the start condition at least according to the raw material quantity parameter. If so, it generates a start signal and sends it to the mixing module; after receiving the start signal, the mixing module starts the rotation assembly to perform mixing for a predetermined time and at a predetermined speed; the operation module is also communicatively connected to the detection unit to receive the detection parameter and controls the reaction process according to the detection parameter. The mixing in this embodiment refers to controlling the rotation assembly by the mixing module to drive the tank body 31 to rotate or drive the rotatable stirring blades 35 in the tank body 31 to rotate to achieve the effect of mixing the reaction solution. The fully automatic in vitro biosynthesis reaction system of the present application can realize the full process automation of the feeding, stirring, material taking, and cleaning processes, reducing the workload and operation knowledge requirements of the operators; at the same time, it can also control the reaction process according to the real-time detection parameters, saving time and effort to achieve the full process quality control.

[0068] In one embodiment, a plurality of protruding structures are uniformly arranged on the inner wall of the tank body 31; and / or a plurality of stirring blades 35 are arranged in the tank body 31, and the stirring blades 35 can be rotatable or non-rotatable; the non-rotatable stirring blades 35 are arranged at intervals along the inner peripheral wall of the tank body 31. Preferably, a plurality of flow holes are arranged on the stirring blades 35, and the distance between adjacent flow holes is equal. Specifically, the main material of the tank body 31 is stainless steel, and 3-6 stirring blades 35 are provided. The stirring blades 35 are integrally formed as a whole, with a smooth surface, and are fixed to the tank wall by welding. The welds are polished to improve the surface smoothness, reduce the resistance to the flow of the reaction solution, accelerate the mixing of the reaction solution, and improve the reaction efficiency. As Figure 9 shown, a plurality of flow holes are arranged on the contour surface of each stirring blade 35, so that the reaction solutions in the regions divided by the stirring blades 35 can interact through these flow holes, improving the mixing effect. For better mixing efficiency, the loading height of the reaction solution should not be too high, generally ensuring that the height of the stirring blades 35 is 1-100 mm higher than the reaction solution.

[0069] In one embodiment, the rotating assembly includes: a large gear 38, a small gear, and a driving device. The large gear 38 is radially and circumferentially fixed outside the tank body 31 of the reaction tank through a gear connecting plate 37; the small gear meshes with the large gear 38 and is arranged on the rotating shaft of the driving device; the driving device rotates the small gear to complete the hybrid drive. Specifically, as Figure 4 , 5 shown, the tank body 31 is each provided with a sealing cover assembly at the front and the rear. The rear sealing cover assembly is penetrated by a slip ring 39 and includes a tail sealing plate 36 that seals the rear end cover of the tank body 31 and a gear connecting plate 37 that connects and fixes the large gear 38 and the tail sealing plate 36. A slip ring connecting piece 361 is also provided in the middle of the tail sealing plate 36 to connect the slip ring 39. The large gear 38 is located at the outermost end of the rear sealing cover assembly.

[0070] In one embodiment, the large gear 38 and the small gear are helical gears; preferably, the helical gears are 15° inclined gears. The large gear 38 and the small gear, as meshing helical gears, have opposite helix directions, that is, one is a left-handed helical gear and the other is a right-handed helical gear. The helical gears are 15° inclined gears. The tooth line of the helical gear winds along the cylinder and is a kind of cylindrical gear. Classified by the gear shaft, the helical gear belongs to the parallel shaft gear. Helical gear transmission has the advantages of good meshing performance, large contact ratio, and high transmission accuracy, because helical gears can better eliminate the influence of pitch error on accuracy. The principle is that the torque fluctuation caused by friction is smaller. When spur gears mesh, it is a straight-line to straight-line contact, and when helical gears mesh, it is a curve to curve contact. Therefore, the principle of the effective friction is different. Compared with spur gears, helical gear transmission has higher accuracy and also has advantages such as high strength or low noise / vibration, which can ensure the more stable operation of the transmission system.

[0071] In one embodiment, the reaction raw material quantity feedback unit has a weighing sensor arranged at the bottom of the tank body 31, and the weighing sensor is communicatively connected to the operation module. Specifically, during feeding, the operation module reads the current equipment weight and the weight before feeding from the weighing sensor for comparison to determine whether the current raw material quantity parameter reaches the preset feeding quantity; during discharging, similarly, the operation module reads the current equipment weight and the weight before discharging from the weighing sensor for comparison to determine whether the current raw material quantity parameter reaches the preset discharging quantity.

[0072] In one embodiment, the detection unit includes: a reaction condition detection unit and / or a reaction liquid parameter detection unit. The reaction condition detection unit includes: a reaction liquid temperature detection component that obtains a reaction liquid temperature parameter by detecting the reaction liquid temperature and / or a gas detection component 4 that obtains an ethanol gas concentration parameter by detecting the concentration of ethanol gas generated during the reaction in the tank body; the reaction liquid parameter detection unit includes: a liquid detection component 5 that obtains a reaction liquid physical and chemical parameter by detecting the reaction liquid physical and chemical parameter and / or an on-line quantitative detection device 7 for reaction products that detects the content of reaction products generated in the reaction liquid to obtain a reaction product content parameter.

[0073] In another embodiment, the operation module further determines whether the start-up conditions are met according to the reaction solution temperature parameter and / or the ethanol gas concentration parameter. Taking in vitro protein synthesis as an example, the biological reaction solution for in vitro biosynthesis is extremely sensitive to temperature. Usually, the protein raw materials are stored in an environment of -18°C, and after being mixed into the reaction solution, low-temperature storage is also required. The reaction of the biological reaction solution is promoted by heating. However, if the temperature is too high, the protein will be inactivated, and if the temperature is too low, the reaction efficiency will be affected, both of which will affect the activity of the reaction solution. At present, the manual detection of the reaction solution temperature is restricted by human factors, and the detection result of the reaction solution temperature cannot be updated in real time, which is not conducive to improving the reaction efficiency and controlling the reaction quality. Ethanol gas is released during the reaction of the biological reaction solution. If the concentration of ethanol gas is too high, it will inhibit the protein reaction. Therefore, during the production process, the reaction solution needs to be stirred to fully react and precipitate ethanol gas. To sum up, ensuring that the ethanol gas concentration in the reaction solution is below the safety threshold and ensuring that the temperature of the reaction solution does not exceed the preset temperature threshold range are the keys to improving the quality of the reaction solution for most biological reaction solution stirring reactions. The operation module of the present application further determines whether the start-up conditions are met according to the reaction solution temperature parameter and / or the ethanol gas concentration parameter, which is conducive to strictly controlling the reaction quality at the initial stage and improving the reaction efficiency.

[0074] Specifically, in one embodiment, the determination of the start-up conditions includes: whether the raw material quantity parameter reaches the preset feed quantity, whether the reaction solution temperature parameter is within the preset temperature threshold range, and whether the ethanol gas concentration parameter exceeds the upper limit of the preset ethanol gas concentration threshold. All the above conditions must be met simultaneously to generate a start signal and send it to the hybrid module.

[0075] In one embodiment, the fully automatic in vitro biosynthesis reaction system of the present application can also automatically manage various parameters during the production process, enabling one-key production, further simplifying the operation. At the same time, the operator can first conduct a trial production, then save the parameters, and perform one-key production, realizing the free switching from sample trial production to mass production of products.

[0076] In one embodiment, the operation module further determines the activity of the reaction solution according to the physicochemical parameters of the reaction solution: when the physicochemical parameters of the reaction solution do not exceed the preset activity range, the activity of the reaction solution is valid and the reaction continues; when the physicochemical parameters of the reaction solution exceed the preset activity range, the activity of the reaction solution fails; further, when the duration of the failure of the activity of the reaction solution does not exceed the preset failure time range, the reaction is not failed and the reaction continues; if it exceeds, the reaction fails.

[0077] Specifically, the reaction activity is the most crucial criterion for judging in vitro biosynthesis reactions. Therefore, according to the type of in vitro biosynthesis reactions, it is very crucial to select appropriate physicochemical parameters of the reaction solution as the criterion for judging the activity of the reaction solution. The judgment of reaction activity can select at least one physicochemical parameter according to different target products, that is, a single physicochemical parameter can be selected or multiple physicochemical parameters can be selected. Each physicochemical parameter has a preset activity range corresponding to different target products. Therefore, effective activity means that each physicochemical parameter index of the reaction solution corresponding to the preset activity range needs to be within its corresponding preset activity range. For example, the preset activity ranges are pH value A, conductivity value B, fluorescence dissolved oxygen value C, and turbidity value D, corresponding to 4 different physicochemical parameters respectively. Only when each physicochemical parameter of the reaction solution corresponding to the preset activity range meets its respective preset activity range is the reaction solution activity effective. Otherwise, as long as one of them does not meet the range, it is considered that the reaction activity fails. Further, when judging that the reaction solution activity fails, if the preset reaction solution activity failure time range is no more than 500 seconds, then when the duration of the reaction solution activity failure does not exceed 500 seconds, the operation module controls the reaction to continue; when the duration of the reaction solution activity failure exceeds 500 seconds, the operation module judges that the reaction fails and controls the reaction to terminate.

[0078] In one embodiment, the operation module further regulates the operation conditions according to the reaction liquid temperature parameter and / or the ethanol gas concentration parameter: when the reaction liquid temperature parameter and / or the ethanol gas concentration parameter do not conform to the preset temperature range and / or the preset ethanol concentration range, the operation module activates the corresponding temperature control component and gas regulation component; further, when the duration of non-conformance does not exceed the corresponding preset automatic recovery time range for temperature non-conformance and / or the preset automatic recovery time range for ethanol concentration non-conformance, the reaction continues; when the duration of non-conformance exceeds the corresponding preset automatic recovery time range for temperature and the preset automatic recovery time range for gas concentration, the reaction stops; furthermore, when the duration of non-conformance does not exceed the corresponding preset stop time range for temperature non-conformance and / or the preset stop time range for ethanol concentration non-conformance, the reaction resumes automatically and continues; when the duration of non-conformance exceeds the corresponding preset stop time range for temperature non-conformance and / or the preset stop time range for ethanol concentration non-conformance, it is necessary to determine whether the physical and chemical parameters of the reaction liquid conform to the preset activity range. If they conform, the reaction continues; if not, the reaction fails. Specifically, for example, if the preset automatic recovery time range for temperature non-conformance and / or the preset automatic recovery time range for gas concentration non-conformance are both no more than 3 seconds, when the duration of the above non-conformance is less than 3 seconds, the operation module controls the reaction to continue; when the duration of the above non-conformance exceeds 3 seconds, the operation module controls the reaction to stop. If the preset stop time range for temperature non-conformance and / or the preset stop time range for gas concentration non-conformance are both no more than 30 seconds, when the duration of the above non-conformance does not exceed 30 seconds, the operation module controls the reaction to resume automatically; when the duration of the above non-conformance exceeds 30 seconds, the operation module also needs to determine whether the physical and chemical parameters of the reaction liquid conform to the preset activity range. If they conform, the reaction continues; if not, the reaction fails.

[0079] In one embodiment, the reaction liquid temperature detection component includes a reaction liquid temperature sensor disposed on the feed pipe 8 near the tank body. The reaction liquid temperature sensor sends the reaction liquid temperature parameter to the operation module in real time to determine whether temperature control adjustment is required. Specifically, the reaction liquid temperature sensor is placed on the pipeline closest to the tank body 31 to minimize the temperature change caused by external heat exchange after the reaction liquid comes out of the tank body 31, ensuring measurement accuracy. Additionally, Figure 6 As can be seen, during the reaction process, since the liquid detection component 5 is always working, the reaction liquid in the tank body 31 is continuously pumped out of the tank body 31 and transported to the closed detection box body 51. The part of the feed pipe 8 extending into the tank body 31 is also borrowed for use as the liquid extraction pipe of the liquid detection pipeline 54. That is, the reaction liquid temperature sensor near the tank body 31 is actually located at the part where the liquid extraction pipe of the liquid detection pipeline 54 and the feed pipe 8 share the part extending into the tank body 31. Therefore, the reaction liquid temperature sensor can always transmit accurate reaction liquid temperature parameters.

[0080] In one embodiment, the gas detection assembly 4 includes: a large ventilation tube 41, a suction tube 44, and an ethanol gas concentration detection device; Figure 7 As shown, the first end of the large ventilation tube 41 extends into the tank body 31 through the closed bearing 42, and the port at the second end of the large ventilation tube 41 is located outside the tank body 31; the exhaust pipe 44 is partially nested and installed in the large ventilation tube 41, one end of which extends out of the tank body 31 from the port at the second end of the large ventilation tube 41 to be connected to the ethanol gas concentration detection device, and the other end of which passes through the first end of the large ventilation tube 41 and is located in the tank body 31; preferably, the exhaust pipe 44 extends upward and passes through the first end; the ethanol gas concentration detection device extracts the gas in the tank body through the exhaust pipe 44 and inputs it into the ethanol gas concentration detection device to detect the ethanol gas concentration in the extracted gas; the ethanol sensor 471 in the ethanol gas concentration detection device sends the ethanol gas concentration parameter to the operation module to determine whether to adjust the ethanol gas concentration. Specifically, the large ventilation tube 41 is firstly fixed in the hollow of the slip ring 39 by the sealed bearing 42. When the tank body 31 is self-stirred and turned over, the large ventilation tube 41 is stationary, and the air extraction pipe 44 and a part of the feed pipe 8 that are arranged in the large ventilation tube 41 are also stationary. The large ventilation tube 41 is constantly exchanging air inside and outside the tank body 31, and the air extraction pipe 44 extends upward in the tank body 31, thereby avoiding the inaccurate detection result of the ethanol gas concentration detection device caused by directly extracting the air that has just been exchanged into the tank body 31.

[0081] In one embodiment, Figure 8 As shown, the ethanol gas concentration detection device includes a suction component 49, a first suction pipe 46, a transfer detection chamber 47, and a second suction pipe 48 connected in sequence; wherein, the transfer detection chamber 47 includes a detection chamber and an ethanol sensor 471, and the gas extracted from the suction pipe 44 is introduced from the second suction pipe 48, and the gas detected by the detection chamber is discharged through the discharge point, preferably, discharged through the suction component 49. Specifically, the part other than the ethanol concentration detection probe of the ethanol sensor 471 is sealed and separated outside the detection chamber by using a sealing gasket 472 at the opening of the detection chamber. The suction component 49 is generally a suction motor, which is also controlled by the operation module. In the process of the gas in the tank body 31 being drawn into the closed transfer detection chamber and discharged, the gas in the transfer detection chamber is detected, which can avoid the problem of low detection accuracy caused by open sampling. At the same time, the suction pipe 44 can also accelerate the discharge of the gas in the tank body 31 through the suction component 49, and reduce the ethanol gas concentration below the safety threshold as much as possible.

[0082] In one embodiment, Figure 9As shown in the figure, the liquid detection component 5 includes: a sealed detection box body 51, multiple physical and chemical sensors 52, and a liquid detection pipeline 54; the detection box body 51 is used to hold the reaction liquid from the tank body as a detection sample, and the probe of the sensor 52 is inserted to detect the detection sample; the liquid detection pipeline 54 communicates with the detection box body 51 and the tank body 31 to form a circulating liquid detection path, and the detection sample enters the detection box body 51 from the liquid detection pipeline 54 and then returns to the tank body 31; the physical and chemical sensors 52 send the physical and chemical parameters of the reaction liquid to the operation module to judge whether to pause, continue or end the hybrid drive. Specifically, the liquid detection pipeline 54 further includes: a liquid extraction pipe and a liquid return pipe, a feed three-way valve and a discharge three-way valve, a liquid extraction pump and a liquid return pump. Among them, the feed three-way valve is respectively connected to the liquid extraction pipe, the reaction liquid raw material tank, and the feed pipe 8 of the reaction tank body 31, and the discharge three-way valve is respectively connected to the liquid return pipe, the reaction liquid finished product tank, and the discharge pipe of the reaction tank body 31; the liquid extraction pipe samples from the reaction tank body 31 through the liquid extraction pump, and the liquid return pipe extracts the detection sample in the detection box body 51 through the liquid return pump.

[0083] In one embodiment, as Figure 9 shown, the liquid detection component 5 further includes: a float switch 53, the float switch 53 is arranged in the detection box body 51, and when the liquid entering the detection box body 51 reaches the safety liquid level, the float switch 53 is triggered to close the liquid extraction pump and simultaneously open the liquid return pump. The safety liquid level is the preset highest liquid level. Beyond this safety liquid level, the detection box body 51 is easily overflowed by the detection sample. According to the flow rate of the liquid return pump and the volume of the reaction liquid that can be accommodated in the detection box body 51, the operation time of the liquid return pump can be preset in the corresponding controller. After reaching the operation time, the liquid return pump is closed to make the liquid level in the detection box body 51 reach the working liquid level, and the liquid extraction pump is opened again to extract the liquid. The working liquid level is the lowest liquid level that satisfies the operation of each sensor.

[0084] In one embodiment, the sensor 52 is a dissolved oxygen sensor, a pH value sensor, a turbidity sensor, a conductivity sensor, a reaction liquid concentration sensor, a reaction liquid temperature sensor, a reaction liquid flow sensor or a liquid level sensor. Specifically, the sensor 52 can provide the dissolved oxygen parameter, pH value parameter, turbidity parameter, conductivity parameter, reaction liquid concentration parameter, reaction liquid flow parameter, etc. of the reaction liquid. The operation module judges the activity of the reaction liquid according to the single or multiple physical and chemical parameters of the reaction liquid and their preset corresponding range values. For example, four physical and chemical parameters of the reaction liquid, namely the preset dissolved oxygen parameter, pH value parameter, turbidity parameter, and conductivity parameter, are used as the judgment indexes of the reaction liquid activity, and the appropriate ranges of the above parameters are specified.

[0085] In one embodiment, as Figures 12 - 13As shown in the figure, the on-line quantitative detection device 7 for reaction products includes: a detection chamber 74, an optical device 72, a detection device 71, a sample flow pipeline 76, and a sample detection tube 78; a detection port is provided on the detection chamber 74; the optical device 72 includes a light source 75, the light source 75 is located in the detection chamber 74, and the light source 75 is used to excite the reaction solution from the tank body 31 to generate the light to be detected; the sample detection tube 78 is used to accommodate the reaction solution, at least part of the sample detection tube 78 is placed in the detection chamber 74 and this at least part is a transparent part, and the reaction solution is excited by the light source 75 in this transparent part to generate the light to be detected; the detection device 71 collects the light to be detected through the detection port and converts it into an electrical signal and transmits it to the image processing module in the detection device 71, and the control device of the in-vitro biosynthesis reaction system has a relevant module that converts and calculates the electrical signal to obtain the content parameter of the reaction product in the reaction solution and displays it in real time for the user to timely master the concentration of the reaction product.

[0086] In one example, the quantitative module gives the reaction product content parameter to the operation module to judge whether to pause, continue or end the mixing; the sample flow pipeline 76 has a liquid inlet section and a liquid outlet section 77 that are respectively communicated with the sample detection tube 778, and the reaction solution enters the sample detection tube 78 from the liquid inlet section and flows through the transparent part, and then the reaction solution is discharged from the liquid outlet section. Specifically, the liquid inlet section and the liquid outlet section 77 of the on-line quantitative detection device for reaction products can also be independently communicated with the reaction tank body 31. For substances (such as proteins, nucleic acids, etc.) that can be excited to generate the light to be detected (such as fluorescence) as synthesis products, or substances that can be excited to generate the light to be detected (such as fluorescence) as synthesis substrates, the concentration of the substances that can be excited to generate the light to be detected in the reaction solution can be detected online in real time, so as to understand the reaction process and regulate the reaction, and solve the problem that the concentration of the above substances cannot be detected in real time in the prior art.

[0087] For fluorescence detection, after the reaction solution is excited by the light source 75, the fluorescent group will emit fluorescence, and then through the optical device 72, an instrument such as an optical camera that can take pictures can take pictures, and the detection device 71 can analyze the concentration of the fluorescent substance in the reaction solution according to the fluorescence situation shown in the image, and then reflect the generation amount of the reaction product. When the light to be detected is Raman light, the optical device 72 further includes an optical path setting 73 that guides the excitation light to excite the reaction solution and collects the Raman light generated by the excited reaction solution, and the detection device 71 has a detector, and the detector can split the collected Raman light through the detection port to convert it into an electrical signal to form a Raman spectrum. There are many optical path settings 73 for realizing Raman spectrum detection in the prior art, which can all be borrowed.

[0088] In one embodiment, a gas disinfection part is further included, the gas disinfection part includes an ultraviolet lamp, the ultraviolet lamp is installed outside the reaction tank assembly and inside the housing assembly 1 surrounding the outside of the reaction tank assembly, and the ultraviolet lamp is controlled by the operation module and remains turned on during the mixing process.

[0089] In one embodiment, as Figure 5 shown, an inspection door 32 is provided on the tank body 31. A door magnetic sensor is installed on the inspection door 32. The door magnetic sensor sends a door magnetic signal to the operation module, and the operation module also determines whether the start condition is met according to the door magnetic signal. Specifically, there is a sealing cover assembly at the front and rear of the tank body 31. The front sealing cover assembly includes: a monitoring door 32 at the forefront, a roller track 34 sleeved on the tank body 31, and an end cover connecting the monitoring door 32 and the roller track 34. In the middle of the monitoring door 32 is a transparent window, and the surface of the transparent window has a convex lens effect with a 5 mm radian.

[0090] In one embodiment, a full-automatic in vitro biosynthesis reaction system provided by the present invention further includes a discharging assembly. The discharging assembly has a discharging pipe communicated with the tank body 31 and a discharging pump communicated with the discharging pipe. Specifically, after the reaction ends, the reaction liquid in the tank body 31 is pumped out of the tank body 31 by the discharging pump through the discharging pipe.

[0091] In one embodiment, a part of the discharging pipe extends into the tank body 31 through a slip ring. One end of the part extending into the tank body 31 extends along the radial direction of the tank body 31 along the inner wall of the tank body 31 towards the circumferential edge, and the other end is connected to the rotor interface of the slip ring 39. Specifically, as Figure 6 shown, the part of the discharging pipe extending into the tank body 31 is also borrowed for use as the return pipe of the liquid detection pipeline 54. The slip ring 39 has at least one passage for transmitting liquid medium, that is, a corresponding interface on each of the rotor and the stator to connect the inside of the rotating tank body 31 and the outside of the stationary tank body 31, thereby effectively solving the problem of external connection of the tank body 31 in the rotating state. When the tank body 31 rotates and flips by itself, the part of the discharging pipe extending into the tank body 31 also flips with the reaction tank 1. To ensure that the tank body 31 can discharge materials smoothly, when the tank body 31 stops flipping, the part of the discharging pipe extending into the tank body 31 should be at the bottom of the tank body 31 to drain the reaction liquid, and a positioning part needs to be set to ensure that the stop angle and position of the tank body 31 meet the requirements. The positioning part is used to sense and position the stop position of the tank body 31, and the positioning part is respectively connected to the hybrid module and the operation module to ensure that the stop angle of the tank body 31 can meet the requirement that the pipe orifice of the part of the discharging pipe extending into the tank body 31 extending towards the circumferential edge is at the bottom of the tank body 31.

[0092] In one embodiment, a full-automatic in vitro biosynthesis reaction system provided by the present invention further includes a circulation temperature control assembly 6, as Figures 5 - 8 shown, the circulation temperature control assembly 6 includes: a coil 61 sleeved on the outer wall of the tank and closely fitted with it, a constant temperature medium liquid tank 64, and a circulation temperature control pipeline 63 connecting the coil 61 and the constant temperature medium liquid tank 64 to form a circulation path of the medium. Specifically, as Figures 3 - 4As shown, the coil pipe 61 is tightly wound around the outer wall of the tank body 31, which belongs to a relatively convenient detachable installation and does not occupy the internal space of the tank body 31, so it will not interfere with the stirring and reaction processes. The circulation temperature control assembly 6 further includes at least one coil pipe pressing strip 62. The coil pipe pressing strip 62 is axially pressed on the coil pipe 61 along the outer wall of the tank body 31 and is adapted to be fixed integrally with the outer wall of the tank body 31. The coil pipe pressing strip 62 can be one or can be fully distributed, and its two ends can be welded integrally with the tank body 31. On the one hand, it can increase the pressing force between the coil pipe 61 and the tank body 31. On the other hand, the coil pipe pressing strip 62 itself can also accumulate a part of heat, which increases the heat conduction area relative to the tank body 31 and improves the heat exchange efficiency. The circulation temperature control assembly 6 further includes a heat insulation material layer, such as sponge 65. The sponge 65 fully covers the outer wall of the coil pipe 61. In one embodiment, a heat insulation material layer is further wrapped on the outer wall of the coil pipe 61 for heat insulation, which can not only shorten the heating time but also save energy. The circulation temperature control assembly 6 further includes a medium circulation pump 66. The medium circulation pump 66 can adjust the flow rate of the medium in the circulation temperature control pipeline 63.

[0093] In one embodiment, the operation module further includes a temperature control adjustment module. The temperature control adjustment module adjusts the set temperature of the constant temperature medium liquid tank 64 according to the obtained reaction liquid temperature parameter to achieve temperature control adjustment. Specifically, the medium in the constant temperature medium liquid tank 64 can be selected from water, heat-conducting oil, silicone oil, ethylene glycol aqueous solution, etc. A medium with a set temperature of T °C (such as 30 °C, and the specific value can be adapted according to the optimal temperature required by the reaction liquid in the tank body 31) circulates between the inside of the coil pipe 61 and the constant temperature medium bath tank 64. Then the coil pipe 61 itself is also heated to T °C. The inner circle of the coil pipe 61 contacts the outer wall of the tank body 31 for heat exchange, so that the tank body 31 is also heated to T °C. Then the reaction liquid in the tank body 31 will also be evenly heated to T °C through heat exchange. It can be seen that the heating process of the reaction liquid is indirect heating through multiple heat exchanges. The reaction liquid does not directly contact active heating devices such as heating pipes, and will not cause local overheating and inactivation of the reaction liquid. Since the flowing medium liquid in the coil pipe 61 has a relatively stable temperature, when it fully covers the outer wall of the tank body 31, the heating will be very uniform, which is beneficial to quickly and efficiently synthesize proteins, thereby improving the protein yield and activity retention rate.

[0094] In one embodiment, the coil pipe 61 is formed by folding a metal pipe in half and then spirally curling it with equal diameter. The coil pipe 61 has two adjacent parallel pipe orifices, which are the coil pipe liquid inlet and the coil pipe liquid outlet respectively. Specifically, the pipeline of the coil pipe 61 is folded back to form two parallel ones. One is connected to the medium outlet, and its temperature is generally higher. The other is connected to the medium inlet, and its temperature is lower due to heat dissipation along the way. Therefore, the coil pipe liquid inlet and the coil pipe liquid outlet are arranged adjacent to each other, and the two pipelines are arranged in parallel together and coiled, which is more helpful to ensure the uniform temperature distribution of the entire coil pipe 61, so as to ensure uniform heating of the reaction liquid.

[0095] In one embodiment, the coil inlet and the coil outlet are respectively communicated with the medium outlet and the medium inlet in the circulation temperature control pipeline 63 through the slip ring 39. Specifically, since the coil 61 is relatively stationary with respect to the tank body 31, in order to rotate with the tank body 31 without causing leakage of the medium liquid, it is necessary to set a slip ring 39 between the stationary part (circulation temperature control pipeline 63) and the moving part (coil 61) with a connection relationship to perform the static-dynamic conversion through the slip ring 39. In this way, the circulation temperature control assembly 6 of this embodiment can adapt to the usage situation where the tank body 31 rotates.

[0096] Taking the production control of a certain protein solution as an example, the control method provided by the present invention will be described in detail:

[0097] (1) Startup: Check the production record. If there is a short-term interruption in production, resume to the state before the interruption and continue to complete the production. Otherwise, enter the normal operation process.

[0098] (2) Preset parameter confirmation: Under the normal operation process, select the production model as "10 liters of a certain protein solution", import the operation data packet corresponding to this production model. Various preset parameters are pre-stored in this operation data packet: for example, the preset feed rate of "10 liters of a certain protein solution" is 10L, the optimal drug temperature is 28 degrees, the preset temperature range is 25 - 30 degrees, the preset reaction stirring speed is 9 RPM, the preset reaction time is 3 hours, the upper limit of the preset ethanol concentration range is 870 PPM, the preset activity range is pH value A, conductivity value B, fluorescence dissolved oxygen value C, turbidity value D, the preset failure time range is not more than 500 seconds, the automatic recovery time range for the preset temperature not meeting the standard is not more than 3 seconds, the automatic recovery time range for the preset ethanol concentration not meeting the standard is not more than 3 seconds, the stop time range for the preset temperature not meeting the standard is not more than 30 seconds, and the stop time range for the preset ethanol concentration not meeting the standard is not more than 30 seconds. Confirm the above preset parameters.

[0099] (3) Preset parameter modification: If the preset parameters of the above production model need to be modified, select custom production, modify the corresponding preset parameters, and after confirming the modification, jump to the liquid inlet process.

[0100] (4) Detection: The detection unit is started while feeding. The detection unit detects various detection parameters from the reaction liquid and / or the gas generated during the reaction process, such as: the reaction liquid temperature parameter obtained by detecting the reaction liquid temperature, the ethanol gas concentration parameter obtained by detecting the ethanol concentration of the gas generated during the reaction, and the reaction liquid activity parameter obtained by detecting the activity of the reaction liquid.

[0101] (5) Feeding: According to the preset feeding volume (10L), the preset liquid inlet pump starts to feed liquid. The remaining liquid inlet volume is queried in real time and the liquid inlet progress is displayed. The current weight of the equipment is read from the weighing sensor and compared with the weight before liquid inlet. It is also necessary to control and adjust the temperature of the medium liquid in the constant temperature medium liquid tank in the circulating temperature control component to increase or decrease according to whether the reaction liquid temperature parameter exceeds the preset temperature range (25°C to 30°C), so as to keep the reaction liquid temperature within 25°C to 30°C; according to whether the ethanol gas concentration parameter exceeds the preset ethanol concentration range (the upper limit is 870PPM), control the exhaust fan or other ventilation equipment in the gas regulation component to increase air replacement to keep the ethanol concentration within 870PPM. When the preset feeding volume is completed and all preset feeding conditions are met, a production feeding completion signal is generated and the process jumps to the reaction process (save the current data to the production record in real time. In case of power failure or other interruptions, after starting up, it will resume to the last data position. The same operation is performed in the following steps).

[0102] (6) Reaction: React according to the preset reaction stirring speed (9RPM) and the preset reaction time (3 hours). After the reaction is completed, control the tank body to stop rotating and generate a reaction completion signal.

[0103] During the reaction, detection parameters are obtained and condition judgments of the detection parameters are carried out, including: whether the reaction liquid activity parameter meets the preset monitoring conditions and whether the reaction liquid temperature parameter and the ethanol gas concentration parameter meet the preset monitoring conditions.

[0104] When judging the reaction liquid activity parameter, when the reaction liquid activity parameter does not exceed the preset activity range (pH value A, conductivity value B, fluorescence dissolved oxygen value C, turbidity value D), the reaction liquid activity is valid and the reaction continues; when the physical and chemical parameters of the reaction liquid exceed the preset activity range, the reaction liquid activity fails; further, when the duration of the reaction liquid activity failure does not exceed the preset failure time range (not exceeding 500 seconds), the reaction is not failed and the reaction continues; if it exceeds, the reaction fails.

[0105] When judging the reaction liquid temperature parameter and / or the ethanol gas concentration parameter, when the reaction liquid temperature parameter and / or the ethanol gas concentration parameter do not meet the preset temperature range (25°C to 30°C) and / or the preset ethanol concentration range (the upper limit is 870PPM), start to regulate the corresponding temperature control component and / or gas regulation component; further, when the non - compliance duration does not exceed the corresponding preset automatic recovery time range for temperature non - compliance (not exceeding 3 seconds) and / or the preset automatic recovery time range for ethanol concentration non - compliance (not exceeding 3 seconds), the reaction continues; when the non - compliance duration exceeds the corresponding preset automatic recovery time range for temperature (not exceeding 3 seconds) and / or the preset automatic recovery time range for gas concentration (not exceeding 3 seconds), the reaction stops;

[0106] Further, when the duration of non-compliance does not exceed the corresponding preset temperature non-compliance stop time range (not exceeding 30 seconds) and / or the preset ethanol concentration non-compliance stop time range (not exceeding 30 seconds), the reaction resumes automatically and continues; when the duration of non-compliance exceeds the corresponding preset temperature non-compliance stop time range (not exceeding 30 seconds) and / or the preset ethanol concentration non-compliance stop time range (not exceeding 30 seconds), it is necessary to determine whether the reaction liquid activity parameter meets the preset activity range. If it meets, the reaction continues; if not, the reaction fails.

[0107] (7) Product taking: Control the product taking pump to discharge the reaction product from the tank through the product taking pipe according to the reaction completion signal. The liquid taking volume can be selected (not selecting the default all remaining liquid volume), and a product taking completion signal is generated after the product taking is completed.

[0108] (8) Cleaning: Turn off the temperature control component and the gas condition component, and start the automatic cleaning program. The automatic cleaning operation is divided into a separate cleaning liquid inlet running time of a preset number of seconds, a cleaning liquid inlet stirring and liquid taking common running time, and a separate cleaning liquid taking running time. Execute the automatic cleaning program in sequence to clean the reaction equipment.

[0109] (9) Others: Update the production record, mark the record status as completed or failed, and the single production process ends.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A control method for realizing in vitro biosynthesis, characterized in that, it includes: After startup, the detection unit continuously detects to obtain detection parameters; According to the preset feed amount stored, control the feed pump to pump the reaction raw materials into the tank for reaction through the feed pipe, and judge whether to generate a feed completion signal according to the preset feed conditions after the feeding is completed; When meeting the preset feed conditions, generate the feed completion signal, and drive the tank to rotate for reaction according to the preset reaction time and preset reaction speed; During the reaction process, continuously judge whether it meets the preset monitoring conditions according to the detection parameters; When not meeting the preset monitoring conditions, select the corresponding preset actions; When the rotation reaches the preset reaction time, control the tank to stop rotating and generate a reaction completion signal; Control the material taking pump to discharge the reaction product from the tank through the material taking pipe according to the reaction completion signal, and generate a material taking completion signal after the material taking is completed.

2. The control method according to claim 1, characterized in that, The detection parameters are the detection parameters obtained by detecting the reaction solution and / or the gas generated during the reaction, and the detection parameters are any one or more of the following: the reaction solution temperature parameter obtained by detecting the reaction solution temperature, the ethanol gas concentration parameter obtained by detecting the ethanol concentration of the gas generated in the reaction, and the reaction solution activity parameter obtained by detecting the activity of the reaction solution.

3. The control method according to claim 2, characterized in that, The reaction solution activity parameter is any one or more of the pH parameter obtained by detecting the pH of the reaction solution through a pH sensor, the dissolved oxygen parameter obtained by detecting the dissolved oxygen of the reaction solution through a fluorescence dissolved oxygen sensor, the turbidity parameter obtained by detecting the turbidity of the reaction solution through a turbidity sensor, and the conductivity parameter obtained by detecting the conductivity of the reaction solution through a conductivity sensor.

4. The control method according to claim 2, characterized in that, The situations of not meeting the preset monitoring conditions are any one or more of the following: The first type: The duration that at least one of the reaction solution activity parameters does not meet the preset activity range exceeds the preset failure time range. At this time, the corresponding preset actions include generating a reaction failure signal and controlling the tank to stop rotating; The second type: The reaction solution temperature parameter does not meet the preset temperature range. At this time, the corresponding preset actions include starting the circulating temperature control component to perform temperature control adjustment of the reaction solution, and judging whether the duration that the reaction solution temperature parameter does not meet the preset temperature range meets the preset automatic recovery time range for temperature non-compliance. If not, control the tank to stop rotating. If so, control the tank to continue rotating; The third type: The duration that the reaction solution temperature parameter does not meet the preset temperature range exceeds the preset stop time range for temperature non-compliance. At this time, the corresponding preset actions include judging whether the reaction solution activity parameter meets the preset activity range. If not, generate the reaction failure signal and control the tank to stop rotating. If so, control the tank to continue rotating; Fourth: The ethanol gas concentration parameter does not meet the preset ethanol concentration range. At this time, the corresponding preset action is to control the gas regulation component to adjust the gas concentration, and determine whether the duration of the ethanol gas concentration parameter not meeting the preset ethanol concentration range meets the automatic recovery time range of the preset non - compliance of ethanol concentration. If not, control the tank body to stop rotating; if so, control the tank body to continue rotating. Fifth: The duration of the ethanol gas concentration parameter not meeting the preset ethanol concentration range exceeds the stop time range of the preset non - compliance of ethanol concentration. At this time, the corresponding preset action includes determining whether the reaction liquid activity parameter meets the preset activity range. If not, generate the reaction failure signal and control the tank body to stop rotating; if so, control the tank body to continue rotating.

5. The control method according to claim 4, characterized in that, the preset feeding condition is that the reaction liquid temperature parameter meets the preset reaction liquid temperature range and / or the ethanol gas concentration parameter meets the preset ethanol gas concentration range; preferably, when the reaction liquid temperature parameter does not meet the preset reaction liquid temperature range, the tank body starts the circulating temperature control component to perform temperature control adjustment of the reaction liquid; when the ethanol gas concentration parameter does not meet the preset ethanol gas concentration range, the tank body controls the gas regulation component to adjust the gas concentration.

6. The control method according to claim 4, characterized in that, According to the failure reaction signal, control the feeding pump to discharge the reaction product from the tank body through the feeding pipe, and generate the feeding completion signal after the feeding is completed.

7. The control method according to claim 1 or 6, characterized in that, when the feeding completion signal is generated, control the cleaning component to perform cleaning according to the stored automatic cleaning parameters.

8. The control method according to claim 7, characterized in that, the automatic cleaning parameters include: the separate cleaning feeding operation time, the cleaning feeding reaction taking common operation time, or the separate cleaning taking operation time of a preset number of seconds.

9. The control method according to claim 1, characterized in that, the preset feeding amount, the preset reaction time, the preset reaction speed, the preset reaction liquid temperature range, the preset ethanol gas concentration range, the automatic recovery time range of the preset temperature non - compliance, the stop time range of the preset temperature non - compliance, the automatic recovery time range of the preset ethanol concentration non - compliance, and the stop time range of the preset ethanol concentration non - compliance will be stored as an operation data packet corresponding to different target products.

10. The control method according to claim 9, characterized in that, the operation data packet has two modes: pre - storage corresponding to the production model or input storage during use corresponding to custom production. The production model refers to the operation data packet with fixed values under different production processes corresponding to different types of target products. If various parameters under the production model need to be modified, it is necessary to switch to the custom production mode for modification.

11. The control method according to claim 1, characterized in that, it also includes terminating the reaction with one key through an emergency stop button.

12. The control method according to claim 1, wherein, when controlling the feed pump to pump the reaction raw materials into the tank for reaction through the feed pipe, the current weight of the device and the weight before feeding are read from the weighing sensor of the tank for comparison, and the remaining feed amount is queried in real time and the feeding progress is displayed; when controlling the take-out pump to discharge the reaction product from the tank through the take-out pipe, the current weight of the device and the weight before take-out are read from the weighing sensor of the tank for comparison, and the remaining take-out amount is queried in real time and the take-out progress is displayed.

13. An in vitro biosynthesis system, wherein, it is used to implement the in vitro biosynthesis control method described in claims 1-12.

14. The system according to claim 13, wherein, it includes: a tank assembly, a rotation assembly, a feeding assembly, a reaction raw material amount feedback unit, a detection unit, and a control device communicatively connected to the foregoing components. Among them, the tank assembly has a tank for accommodating the reaction solution for in vitro biological reaction; the rotation assembly is used to achieve the mixing of the reaction solution; the feeding assembly has a feed pipe connected to the tank and a feed pump connected to the feed pipe; the reaction raw material amount feedback unit is used to detect the raw material amount in the tank to obtain a raw material amount parameter; the detection unit is used to detect the reaction solution and / or the gas generated by the reaction to obtain a detection parameter; the control device has a feeding module, an operation module, and a mixing module; among them, the feeding module controls the feed pump to pump the reaction raw materials into the tank through the feed pipe; the operation module obtains the raw material amount parameter and determines at least according to the raw material amount parameter whether it meets the start condition. If so, a start signal is generated and sent to the mixing module; after receiving the start signal, the rotation module starts the rotation assembly to rotate for a predetermined time and at a predetermined speed; the operation module is also communicatively connected to the detection unit to receive the detection parameter and control the reaction process according to the detection parameter.

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