In-vitro biosynthesis reaction apparatus and in-vitro biosynthesis method

Abstract

The application provides a reaction device for in-vitro biosynthesis and an in-vitro biosynthesis method to solve the problem that in-vitro biosynthesis cannot be expanded at present, wherein the reaction device for in-vitro biosynthesis is characterized by comprising: a reaction body, the reaction body is used for accommodating a protein synthesis reaction solution to carry out in-vitro biosynthesis reaction, the reaction body is provided with an opening part, the opening part has a feeding port for allowing the synthesis reaction solution to enter the reaction body and a discharging port for discharging the product after reaction; and a separation part, the separation part is used for incompletely separating the internal space of the reaction body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a reaction apparatus and method for in vitro biosynthesis. Background Technology

[0002] Various in vitro biosynthetic methods refer to synthesis in contrast to in vivo synthesis. For example, in vitro cell-free protein synthesis mainly relies on cell-free expression systems. It uses exogenous target DNA as a template for protein synthesis and artificially controls the addition of substrates and cofactors related to transcription and translation, etc., to achieve in vitro synthesis of the target protein through reaction solutions.

[0003] However, currently, in vitro synthesis is carried out on a small scale in the laboratory, and there is no production equipment that can be scaled up.

[0004] There is an urgent need to develop a production scale-up device that can significantly increase the volume while maintaining the maximum reactivity. Summary of the Invention

[0005] The present invention aims to solve the problem that current in vitro biosynthesis cannot be scaled up for production. To this end, the present invention provides the following technical solution.

[0006] This invention provides a reaction apparatus for in vitro biosynthesis, characterized in that it comprises:

[0007] The reaction chamber body is used to contain the synthesis reaction solution for in vitro biosynthesis. The reaction chamber body is provided with an opening, which has an inlet for the synthesis reaction solution to enter the reaction chamber body and an outlet for the reaction product to be discharged. The partition is used to partially divide the internal space of the reaction chamber body.

[0008] The reaction apparatus provided by the present invention also has the following features: wherein the reaction chamber body is a tank, preferably a horizontal tank, and the tank is spherical or ellipsoidal.

[0009] The reaction apparatus provided by the present invention also has the following feature: wherein the partition includes at least one partition element, preferably, the partition element is sheet-shaped.

[0010] The reaction apparatus provided by the present invention also has the feature that multiple separators are evenly spaced.

[0011] The reaction apparatus provided by the present invention also has the following feature: wherein the length of the separator extends in a curved or broken line.

[0012] The reaction apparatus provided by the present invention also has the following feature: wherein the line formed between the two ends of the length of the separator has an included angle α with the central axis of the internal space of the reaction chamber body which is in the same direction as the length extension direction. Preferably, the included angle α is less than or equal to 60°, more preferably less than or equal to 45°, and even more preferably less than or equal to 20°.

[0013] The reaction apparatus provided by the present invention also has the following feature: at least one partition is rotatably and / or non-rotatably fixed on the inner wall of the reaction chamber body.

[0014] The reaction apparatus provided by the present invention also has the feature that, when the partition is disposed on the inner wall, one side of the sheet-like partition faces the inner wall of the reaction chamber body.

[0015] The reaction apparatus provided by the present invention also has the following feature: when the partition is disposed on the inner wall, one side of the sheet-like partition faces the inner wall of the reaction chamber body, while a recessed area is provided on the other side of the partition away from the inner wall.

[0016] The reaction apparatus provided by the present invention also has the feature that when one side of the sheet-like separator faces the inner wall of the reaction chamber body, the angle between the sheet-like surface of the separator and the opposite inner wall is not equal to 90°.

[0017] The reaction apparatus provided by the present invention also has the following feature: in use, the reaction is carried out by rotating the reaction chamber body, preferably by deviating the horizontal central axis of the reaction chamber body from the horizontal state.

[0018] The reaction apparatus provided by the present invention further has the following feature: at least one flow hole is provided on the separator. Preferably, the flow holes on the at least one separator are uniformly distributed.

[0019] The reaction apparatus provided by the present invention further has the following feature: when the separator is disposed on the inner wall, there is at least one discontinuity between the separator and the inner wall. Preferably, the discontinuities corresponding to the at least one separator are equidistantly distributed.

[0020] The reaction apparatus provided by the present invention also has the following features: the opening is further provided with a ventilation unit, the ventilation unit having an air inlet and / or an exhaust port, the air inlet being used to allow external gas to enter the internal space of the reaction chamber body, and the exhaust port being used to allow gas in the internal space to be discharged. Preferably, one or more of the feed inlet, discharge outlet, air inlet and exhaust port are the same opening.

[0021] The reaction apparatus provided by the present invention also has the following features: an air intake unit and / or an exhaust unit, wherein the air intake port allows outside gas to enter the internal space through the air intake unit, and the exhaust port allows gas in the internal space to be discharged from the exhaust unit through the exhaust unit.

[0022] The reaction apparatus provided by the present invention also has the following features: wherein the air intake unit includes at least one air intake pipe, one end of which faces the air inlet and supplies outside gas to the internal space, or one end of which extends from the air inlet into the internal space. In this case: preferably, at least one air intake pipe extends along the horizontal direction of the reaction chamber body; more preferably, at least one air outlet is provided on the pipe wall of the portion of the at least one air intake pipe that enters the internal space; preferably, at least one air outlet is evenly distributed, and / or one end of the air intake pipe that enters the internal space is sealed.

[0023] The reaction device provided by the present invention also has the following features: the exhaust unit includes an exhaust pipe, preferably, a portion of the intake pipe is disposed in the exhaust pipe between the two ends, one end of the intake pipe extends out from one end of the exhaust pipe and into the internal space, and the other end of the intake pipe extends out from the other end of the exhaust pipe and is connected to the gas source.

[0024] The reaction apparatus provided by the present invention further comprises: a cover assembly having a cover adapted to the feed inlet for closing the feed inlet. Preferably, the cover assembly further comprises a sealing ring, a lid latch, and a locking handle.

[0025] The reaction apparatus provided by the present invention also has the following feature: when a cover assembly is included, the venting unit further includes a vent hole, which is disposed on the cover, and the venting pipe is connected to the internal space through the vent hole.

[0026] The reaction apparatus provided by the present invention also has the following features: a rotating part for rotating the reaction chamber body, preferably, the rotating part rotates the reaction chamber body by being rotatably connected to the reaction chamber body, and / or the rotating part is further provided with a rotating wheel, the reaction chamber body is disposed on the rotating wheel, and the rotating wheel rotates with the reaction chamber body.

[0027] The reaction apparatus provided by the present invention also has the following feature: the rotating part includes a driving unit, which drives the reaction chamber body to rotate.

[0028] The reaction apparatus provided by the present invention also includes: a temperature control device for maintaining the reaction under predetermined temperature conditions; preferably, the temperature control device is used to control the temperature of the gas entering the reaction chamber body to maintain the predetermined temperature conditions.

[0029] The reaction apparatus provided by the present invention further comprises: a housing, which is adapted to the reaction chamber body to accommodate the reaction chamber body.

[0030] Preferably, the housing has a combination of any one or more of the following features:

[0031] (1) An opening and closing door is provided on the housing, preferably, a vent is provided on the opening and closing door;

[0032] (2) A display screen is provided on the outside of the housing, preferably the display screen is located on the outer surface of the top of the housing;

[0033] (3) A shelf for placing items is provided on the outside of the shell. Preferably, the surface of the shelf is provided with anti-slip texture. More preferably, the outer surface of the top of the shell is the shelf.

[0034] (4) The shell is provided with any one or more of the following: an air inlet channel for allowing external gas to enter the internal space of the reaction chamber body, an exhaust channel for allowing gas to exit the internal space of the reaction chamber body, a feed channel for allowing the reaction liquid for synthesis to enter the internal space of the reaction chamber body for in vitro biosynthesis, and an outlet channel for allowing the product after the reaction to exit.

[0035] The present invention also provides an in vitro biosynthesis method, characterized in that: the aforementioned reaction apparatus is used to contain the reaction solution for synthesis to carry out the in vitro biosynthesis reaction.

[0036] The in vitro biosynthesis method provided by the present invention also has the following feature: wherein the volume of the synthesis reaction solution used for the reaction accounts for less than or equal to 30% of the volume of the reaction chamber body.

[0037] The in vitro biosynthesis method provided by the present invention also has the following characteristics: in the process of reaction, an external gas is introduced into the internal space, and / or the gas in the internal space is discharged.

[0038] The in vitro biosynthesis method provided by the present invention also has the following feature: during the reaction process, the reaction chamber body of the reaction device is rotated, preferably at a speed of less than or equal to 120 r / min.

[0039] Invention Function and Effect

[0040] The in vitro biosynthesis reaction apparatus and in vitro biosynthesis method provided by this invention have at least the following functions and effects:

[0041] (1) By partially separating the internal space of the reaction chamber body through the partition, when the reaction chamber body contains the reaction liquid for in vitro biosynthesis reaction, the reaction liquid in the "flow state" can flow from one space area to another space area, while the reaction liquid flowing through the partition will be blocked by the partition, which increases the mixing intensity of the reaction liquid in the in vitro biosynthesis reaction process, thereby improving the reaction efficiency. Moreover, the overall structure is simple and can be made of stainless steel, which is convenient for large-scale manufacturing and production.

[0042] (2) During the reaction, the reaction chamber body is rotated so that the reaction liquid can be effectively spread on the inner wall, thereby increasing the efficiency of in vitro biosynthesis;

[0043] (3) When the reaction chamber body is a tank, compared with other shapes such as a cuboid, the inner wall has an arc and no dead corners, so it is not easy to generate foam in the reaction, which ensures the quality of synthesis. Moreover, compared with other shapes, the same volume has a larger inner wall surface area, which can spread the reaction liquid thinner, which is more conducive to synthesis.

[0044] (4) The separator is sheet-like, which increases the contact area of ​​the reaction liquid and thus increases the degree to which the reaction liquid is spread thinly;

[0045] (5) The partition is a curved or broken line extension. In the same internal space, compared with a straight line, the contact area of ​​the reaction liquid is increased, which is beneficial to the spread of the reaction liquid. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the overall structure of the reaction apparatus according to Embodiment 1 of the present invention;

[0047] Figure 2 In response to Figure 1 A diagram from another perspective;

[0048] Figure 3 This is a schematic diagram of the reaction chamber body structure of the reaction device according to Embodiment 1 of the present invention;

[0049] Figure 4 This is a schematic diagram showing only the partition section of the reaction apparatus according to Embodiment 1 of the present invention;

[0050] Figure 5 This is a schematic diagram illustrating the isolation of the internal space in Embodiment 1 of the present invention;

[0051] Figure 6 This is a schematic diagram of the structure of the reaction chamber body, whose internal space is not completely separated by a partition, according to Embodiment 1 of the present invention.

[0052] Figure 7This is a schematic diagram of the structure of a separator in the reaction apparatus according to Embodiment 1 of the present invention;

[0053] Figure 8 This is a schematic diagram of the overall structure of the reaction device involved in Embodiment 2 of the present invention;

[0054] Figure 9 In response to Figure 8 A diagram from another perspective;

[0055] Figure 10 This is an assembly diagram of the air intake unit, exhaust unit, and cover assembly according to Embodiment 2 of the present invention;

[0056] Figure 11 This is a schematic diagram of the overall structure of the reaction device involved in Embodiment 3 of the present invention;

[0057] Figure 12 for Figure 11 A side view;

[0058] Figure 13 This is a diagram showing the results of an experiment related to Embodiment 4 of the present invention. Detailed Implementation

[0059] The specific embodiments of the present invention will be described below with reference to the accompanying drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.

[0060] In this article, the direction of the longer axis of the reaction chamber body is called the "length direction" of the reaction chamber body, and the opposite is called the "width direction". For example, for an ellipsoid, when the major axis is horizontal, this horizontal direction is the length direction.

[0061] The "length direction" referred to in this article refers to roughly equal directions, not absolutely consistent directions. Similarly, the "width direction" also refers to roughly equal directions.

[0062] Example 1

[0063] Figure 1 This is a schematic diagram of the overall structure of the reaction apparatus according to Embodiment 1 of the present invention;

[0064] Figure 2 In response to Figure 1 A diagram from another perspective;

[0065] Figure 3 This is a schematic diagram of the reaction chamber body structure of the reaction device according to Embodiment 1 of the present invention;

[0066] Figure 4This is a schematic diagram showing only the partition section of the reaction apparatus according to Embodiment 1 of the present invention.

[0067] like Figures 1-4 As shown, the in vitro biosynthesis reaction apparatus 100 provided in this embodiment includes: a reaction chamber body 10 and a partition 20.

[0068] The reaction chamber body 10 is used to contain the synthesis reaction solution for in vitro biosynthesis. The reaction chamber body 10 is provided with an opening 10a, which has an inlet for allowing the synthesis reaction solution to enter the internal space 11 of the reaction chamber body for reaction and an outlet for discharging the reaction product. The reaction solution can be poured directly into the inlet or the reaction product can be poured out from the outlet. Alternatively, the reaction solution can be fed and discharged in other ways, such as by a feed pipe conveying the synthesis reaction solution into the internal space 11 from the inlet, or by an outlet pipe discharging the reaction product from the internal space 11.

[0069] In one example, the opening 10a also has a ventilation unit with an air inlet.

[0070] And / or exhaust ports. The air inlet is used to allow external gases to enter the internal space of the reaction chamber, thus introducing oxygen-containing gases, such as air, which is beneficial for in vitro biosynthesis reactions; the exhaust port is used to allow gases in the internal space to be discharged, thus timely removing gases that are detrimental to the reaction, such as ethanol, to avoid affecting synthesis efficiency and quality.

[0071] In one example, one or more of the inlet, outlet, air inlet, and exhaust outlet are the same opening, such as... Figure 1 In this example, the feed inlet, discharge outlet, air inlet, and exhaust outlet are all the same opening, namely opening 10a. In other examples, these four openings may be different openings, or several of them may be the same opening.

[0072] Figure 5 This is a schematic diagram illustrating the isolation of the internal space in Embodiment 1 of the present invention.

[0073] Figure 5 In the diagram, the area covered by the diagonal line represents a dividing section.

[0074] The partition 20 is used to partially partition the internal space of the reaction chamber body 10.

[0075] Combination Figure 5To explain, the circle in the diagram represents a cross-section of the reaction chamber, the interior of the circle represents the internal space, and the arc in the middle of the circle represents the partition. "Incomplete partitioning" is relative to "complete partitioning." "Complete partitioning" means dividing the entire internal space into different, unconnected spaces; that is, the partitioned spaces are completely isolated. For example, using a plate to completely partition along a cross-section of the entire internal space to create two spaces. Figure 5 In the left-middle image, the entire cross-section of the partition completely covers a cross-section of the interior space; while "incomplete partitioning" means not forming a complete partition of the interior space, such as... Figure 5 As shown in the middle right figure, although the left, right, and lower edges of the partition contact the inner wall, the upper edge does not. This incomplete partition creates interconnected spatial regions, as shown in the figure below. Figure 5 As shown in the middle right figure, in the cross-section, the part covered by the partition is a spatial region, and the spaces are interconnected through the blank part above.

[0076] Thus, since the separation is incomplete, when the aforementioned reaction liquid is contained, the "flowing" reaction liquid can flow from one spatial region to another, while the reaction liquid flowing through the separation section 20 will be blocked by the separation section 20, thereby increasing the mixing intensity of the reaction liquid during the in vitro synthesis reaction and improving the reaction efficiency.

[0077] Here, "flow state" refers to the reaction liquid not being stationary, but rather undergoing at least partial displacement relative to the inner wall. The reasons for this include, but are not limited to, the following:

[0078] The first type is caused solely by the movement of the reaction chamber body 10, such as vibration or rotation of the reaction chamber body.

[0079] The second type flows only when subjected to other external forces:

[0080] A. For example, it is caused by external agitation, such as the rotation of the partition 20 agitating the reaction liquid and causing the reaction liquid to enter a flowing state;

[0081] B. For example, the reaction liquid can enter a flowing state due to the fluctuation of the reaction liquid caused by the introduction of high-pressure airflow or ultrasonic vibration.

[0082] The third type combines the functions of the first and second types. For example, when the reaction chamber body 10 rotates, the partition 20 also rotates.

[0083] In addition, when the reaction liquid is in a flowing state, the obstruction of the partition 20 also increases the chance of the reaction liquid contacting the inner wall of the reaction chamber body 10. Under certain conditions, such as gravity or rotation, the reaction liquid that comes into contact with the inner wall has the opportunity to be spread thin on the inner wall. The spread reaction liquid has a larger surface area and can contact the air more fully, thereby improving the efficiency of in vitro synthesis.

[0084] In one example, the reaction chamber body 10 is a tank as shown in the figure. Since the tank is spherical or ellipsoidal, it has a larger inner wall surface area compared to other shapes, giving the reaction liquid a greater chance to be spread thinly on the inner wall. Moreover, due to the absence of dead corners, foam is less likely to be generated during the reaction. Preferably, it is a horizontal tank, that is, when the tank is ellipsoidal, the major axis is in the horizontal direction, which avoids the reaction liquid from depositing at the bottom compared to the vertical direction, and is more conducive to spreading.

[0085] In one example, during use, the reaction chamber body 10 is rotated to complete the reaction. This allows the reaction solution to be effectively spread thinly on the inner wall, increasing the efficiency of in vitro synthesis. The degree to which the reaction solution is spread thinly can be adjusted by controlling the volume of the added reaction solution. Preferably, the horizontal central axis of the reaction chamber body 10 is offset from the horizontal state by a certain angle, that is, the reaction chamber body 10 is placed at an angle. This allows the reaction solution to be more easily spread thinly on more of the inner wall when the reaction chamber body 10 rotates.

[0086] Figure 6 This is a schematic diagram of the structure of the reaction chamber body, whose internal space is not completely separated by a partition, according to Embodiment 1 of the present invention.

[0087] In one example, at least one first protrusion 10b is provided on the inner wall of the reaction chamber body 10 (e.g., Figure 6 As shown, the surface area of ​​the inner wall can be increased by the first protrusion 10b, thereby increasing the contact area with the reaction liquid and thus increasing the degree to which the reaction liquid is spread.

[0088] In one example, such as Figure 4 As shown, the partition 20 includes at least one partition 21, thereby partially dividing the internal space 11 to form multiple spatial regions. The multiple partitions 21 can increase the mixing opportunities of the reaction liquid and the opportunity to be spread to the inner wall.

[0089] Figure 7 This is a schematic diagram of the structure of a separator in the reaction apparatus according to Embodiment 1 of the present invention.

[0090] like Figure 7As shown, in one example, the separator 21 is sheet-shaped. This allows the reaction liquid to be spread thinly onto the sheet-shaped surface 22 when it comes into contact with it. This increases the area over which the same volume of reaction liquid can be spread, making it easier to spread thinner and increasing the contact area with air, thus improving reaction efficiency. The sheet-shaped surface 22 here, as... Figure 7 As shown, this is relative to side 23. In this case, the more separators 21 there are, the more surface area they can contact with the reaction liquid, thus increasing the spreading area of ​​the reaction liquid and consequently increasing the overall contact area between the reaction liquid and the air. However, too many separators 21 may result in too short an exposure time for the spread reaction liquid to air, requiring a longer reaction time. Furthermore, too many separators 21 can lead to excessive weight, making it inconvenient to use, while the overall increase in spreading is not significant, resulting in wasted costs.

[0091] In one example, multiple separators 21 are evenly spaced, which can partially divide the internal space 11 into the same spatial regions, so that the spread of the reaction liquid in each spatial region is relatively uniform, that is, the contact area of ​​the reaction liquid is relatively uniform, the reaction efficiency is uniform, and the overall reaction effect is better.

[0092] In one example, the length of the separator 21 extends in a curved or zigzag pattern, which increases the chance of the reaction liquid coming into contact with the separator 21 within the same internal space. This is especially true when the separator is sheet-like, as it increases the contact area and thus improves the synthesis efficiency.

[0093] In one example, such as Figure 6 As shown, the line 21A connecting the two ends of the length of the separator 21 has an angle α with the central axis 11A of the internal space 11 of the reaction chamber body 10, which is in the same direction as the extension of the length.

[0094] The phrase "central axis in the same direction as the length of the extension" means that when the length of the separator 21 extends approximately along the length of the reaction chamber body 10, then the central axis 11A is the central axis in the length direction of the reaction chamber body 10. When the length of the separator 21 extends approximately along the width direction of the reaction chamber body, then the central axis 11A is the central axis in the width direction of the reaction chamber body 10. "In the same direction" means approximately the same, not absolutely the same.

[0095] The angle 'a' here means that the line 21A connecting the two sides is not parallel to the central axis 11A, but rather at an angle, i.e., it is inclined towards the central axis 11A. Preferably, the angle 'a' is less than or equal to 60°, more preferably less than or equal to 45°, and even more preferably less than or equal to 20°. This facilitates the flow of the reaction liquid from one end of the reaction chamber body 10 to the other in the divided spatial regions. Thus, when the end of the separator 21 is connected to an adjacent spatial region, it is convenient to flow from this end to the adjacent spatial region, improving mixing efficiency. The angle 'a' should not be too large, meaning the line 21a should not deviate too much from the axis; otherwise, the reaction liquid will accumulate at one end of the reaction chamber body 10, which is not conducive to thinning.

[0096] In one example, the partition 20 is disposed on the inner wall of the reaction chamber body 10. In practice, it can be rotatably fixed to the inner wall or non-rotatably fixed to the inner wall. When there are multiple partitions 21, they can all be rotatably disposed on the inner wall or partially rotatably disposed on the inner wall. Preferably, when the partition 21 is plate-shaped, one side 23 of the plate-shaped partition 21 faces the inner wall of the reaction chamber body 10, that is, it is rotatably or non-rotatably disposed on the inner wall through the side 23.

[0097] In one example, when one side 23 of the sheet-like separator 21 faces the inner wall of the reaction chamber body 10, the angle between the sheet-like surface of the sheet-like separator and the inner wall opposite (referring to the inner wall where the side 23 is located) is not equal to 90°. In other words, the sheet-like surface is not completely perpendicular to the inner wall, but deviates from the vertical plane by a certain angle b. This relative to the vertical plane can reduce the foam generated in the reaction, improve the reaction effect, and also allow the reaction liquid to be spread more thinly.

[0098] In one example, the length of the separator 21 extends along the length direction of the reaction chamber body 10 and from one end of the reaction chamber body to the other end. That is, the length of the separator is approximately equal to the length of the reaction chamber body, which ensures a larger contact area and thus improves the synthesis efficiency.

[0099] In one example, the separator 21 is provided with at least one flow hole (not shown in the figure), which facilitates the flow of the reaction liquid from one spatial region to another. Preferably, when there are multiple flow holes, the flow holes on at least one separator 21 are evenly arranged on the separator 21. For example, there are three separators 21, and each separator 21 is provided with multiple flow holes. The flow holes on any one or more of the three separators can be evenly arranged.

[0100] In one example, when the separator 21 is disposed on the inner wall, there is at least one discontinuity between the separator and the inner wall, that is, there is at least one gap between the separator 21 and the inner wall, which facilitates the flow of the reaction liquid from one spatial region to another. Preferably, the discontinuities between at least one separator 21 and the inner wall are equidistantly arranged. For example, three separators 21 are disposed on the inner wall, each separator 21 having at least one discontinuity between it and the inner wall, and at least one of the three separators 21 having an equidistant discontinuity between it and the inner wall.

[0101] In one example, such as Figure 6 and 7 As shown, at least one second protrusion 21b is arranged on the outer surface of the separator 21. Similarly, the surface area of ​​the separator 21 can be increased by the second protrusion 21b, thereby increasing the contact area with the reaction liquid and thus increasing the degree to which the reaction liquid is spread.

[0102] In one example, the reaction device 100 also includes an air intake unit. The aforementioned air intake allows outside gas to enter the internal space 11 through this air intake unit.

[0103] In one example, the intake unit 30 includes at least one intake duct, such as Figure 1 As shown, Figure 1 The image shows an air intake pipe 30a, one end of which faces the air inlet and supplies outside gas into the internal space. That is, there is a certain distance between the air intake pipe and the internal space 11.

[0104] In one example, the reaction apparatus 100 also includes an exhaust unit through which the aforementioned exhaust port allows the gas in the internal space 11 to be discharged.

[0105] In one example, the exhaust unit includes at least one exhaust pipe that can discharge gas from the internal space 11 by communicating with or extending into the internal space 11 from the aforementioned exhaust port.

[0106] In one example, the reaction apparatus 100 further includes a rotating part for rotating the reaction vessel.

[0107] The reaction chamber body 10 is preferably rotated by a rotating part that is rotatably connected to it. Preferably, it is rotatably connected to a rotating mounting part located on the opposite side of the opening on the reaction chamber body 10. After the rotatable connection, the opening position of the opening is tilted upwards (e.g., ...). Figure 1 As shown in the figure, this allows the reaction liquid to better contact and spread with the inner wall of the reaction chamber body 10 and the partition 20 during rotation, and prevents the reaction liquid from overflowing even if the opening is large. This makes it easier to feed or discharge when the opening is used as a feed port or discharge port, and ensures a larger air exchange rate when used as an air inlet or outlet port.

[0108] In one example, such as Figure 1 As shown, the rotating part is also equipped with a rotating wheel 62, and the reaction chamber body 10 is mounted on the rotating wheel 62. The rotating wheel 62 rotates with the reaction chamber body 10, which ensures that even if the reaction chamber body 10 has a very large weight, it can be well supported and rotated, making it easier to achieve large-scale in vitro bioreactor production. Figure 1 As shown in the figure, the rotating wheel 62 is mounted on the frame 62a, and the reaction chamber body 10 is mounted on the rotating wheel 62.

[0109] In one example, the rotating part includes a driving unit 63, which drives the reaction chamber body to rotate, such as... Figure 2 As shown, the drive unit 63 drives the reaction chamber body 10 to rotate via a rotating shaft.

[0110] In one example, the reaction apparatus 100 also includes a temperature control device (not shown) for maintaining the in vitro biosynthesis reaction at a predetermined temperature condition, such as by taking measures to keep the reaction chamber body 10 warm, heating or cooling. Preferably, the temperature control device is used to regulate the temperature of the gas entering the reaction chamber body to maintain the predetermined temperature condition, such as by heating the gas entering the internal space.

[0111] In this embodiment, an in vitro biosynthesis method is also provided. This in vitro biosynthesis method uses the aforementioned reaction apparatus 100 to contain the synthesis reaction solution for in vitro biosynthesis reaction. Specifically, the method will be described in conjunction with the aforementioned reaction apparatus 100.

[0112] In one example, in the in vitro biosynthesis method of this embodiment, the volume of the synthesis reaction liquid used for the in vitro biosynthesis reaction accounts for less than or equal to 30% of the volume of the reaction chamber body. This ensures that the reaction liquid is sufficiently thinned and that the reaction efficiency is guaranteed.

[0113] In one example, in the in vitro biosynthesis method of this embodiment, an external gas, such as air, is introduced into the internal space during the in vitro biosynthesis reaction, which is beneficial for the oxygen required for the reaction.

[0114] In one example, in the in vitro biosynthesis method of this embodiment, the gas in the internal space is discharged during the in vitro biosynthesis reaction to prevent the waste gas generated by the reaction from affecting the synthesis.

[0115] In one example, in the in vitro biosynthesis method of this embodiment, during the in vitro biosynthesis reaction process, the reaction chamber body 10 of the reaction apparatus is rotated to allow the reaction liquid to spread thinly on the inner wall and separators, and to be mixed. Furthermore, the rotation speed is less than or equal to 120 r / min, which minimizes foam generation and ensures the quality of the synthesis.

[0116] Example 2

[0117] In this embodiment, the same parts as in Embodiment 1 are given the same drawing numbers, and the same descriptions and explanations are omitted.

[0118] Figure 8 This is a schematic diagram of the overall structure of the reaction device involved in Embodiment 2 of the present invention;

[0119] Figure 9 In response to Figure 8 A schematic diagram from another perspective.

[0120] like Figure 8 and 9 As shown in the figure, there are 6 partitions 21, evenly spaced, and between every two adjacent partitions 21, an identical spatial region 21a is formed.

[0121] In one example, such as Figure 9 As shown, a recessed area 24a is provided on the other side 24 of the sheet-like separator 21 opposite to the inner wall. In this way, when the separator 21 is provided on the inner wall, the reaction liquid can flow between the spatial areas, thereby improving the overall mixing efficiency.

[0122] In one example, such as Figure 8 One end of the intake pipe 30a of the intake unit extends into the internal space 11 from the intake port. In this case, preferably, at least one intake pipe extends along the horizontal direction X of the reaction chamber body, so that the incoming gas can extend as far as possible into the other end of the reaction chamber body 10 that is prone to lack of oxygen gas.

[0123] More preferably, such as Figure 8 At least one air inlet duct 30a is provided with at least one air outlet 31 on the pipe wall of the portion of the air inlet duct 30a that enters the internal space 11. That is, when there are multiple air inlet ducts 30a, each air inlet duct partially enters the internal space. Among these air inlet ducts 30a, at least one air outlet 31 is provided on the pipe wall of the portion of the air inlet duct 30a that enters. Preferably, these air outlets 31 are evenly distributed.

[0124] In one example, when an air vent is provided, one end of the air intake pipe 30a that enters the interior space can be sealed.

[0125] In one example, the exhaust unit includes an exhaust pipe 41, and a portion of the intake pipe 30a is disposed within the exhaust pipe between its two ends. That is, one end of the intake pipe 30a extends from one end of the exhaust pipe 41 into the internal space, and the other end of the intake pipe 30a extends from the other end of the exhaust pipe 41 and connects to the air source. In other words, a portion of the intake pipe 30a is disposed within the exhaust pipe 41. This isolates the intake and exhaust, allowing the intake and exhaust pipes to be located in the same part of the reaction chamber body. Figure 8 As shown, both the air intake and exhaust are located on the left side.

[0126] In one example, the reaction apparatus 100 further includes a cover assembly having a cover adapted to the feed inlet for closing the feed inlet.

[0127] Preferably, the lid assembly further includes a sealing ring, a lid latch, and a locking handle.

[0128] In one example, the exhaust unit also includes an exhaust port 42, which is disposed on the cover 51, and the exhaust pipe 41 is connected to the internal space through the exhaust port 42.

[0129] Figure 10 This is an assembly diagram of the air intake unit, exhaust unit, and cover assembly involved in Embodiment 2 of the present invention.

[0130] In one example, such as Figure 10 As shown, the cover assembly 1, the air inlet unit 30, and the exhaust unit 40 are an integral structure. In use, by inserting one end of the air inlet pipe 31 into the reaction body 10 and the other end of the air inlet pipe 31 extending out of the exhaust pipe 41 to receive the air source, and by fitting the cover 51 with the exhaust hole 42 onto the feed inlet, the entire configuration is completed. The structure is ingenious and easy to install.

[0131] Example 3

[0132] Figure 11 This is a schematic diagram of the overall structure of the reaction device involved in Embodiment 3 of the present invention;

[0133] Figure 12 for Figure 11 A side view.

[0134] like Figure 11 and 12 As shown, in this embodiment, the same components as in Embodiment 1 are designated by the same drawing numbers, and the same descriptions and explanations are omitted. The reaction apparatus 300 in this embodiment also includes a housing 70, which is adapted to the reaction chamber body 10 to accommodate the reaction chamber body 10.

[0135] Preferably, the housing 70 has a combination of any one or more of the following features:

[0136] (1) An opening and closing door 71 is provided on the shell 70. Preferably, a vent hole 71a is provided on the opening and closing door 71, so that the gas inside the reaction chamber body 10 can be discharged or the gas outside can enter the internal space 11.

[0137] (2) A display screen 72 is provided on the outside of the housing 70, that is, it is not inside the housing 70, but on the outside, for example, it is provided on the outer surface. Preferably, the display screen 72 is located on the outer surface of the top 73 of the housing 70, or for example, it is embedded on the outer surface. Through the display screen 72, the reaction process, state and conditions can be displayed, and the reaction and the discharge of reaction products can be completed by touch operation.

[0138] (3) A shelf for placing items is provided on the outside of the shell 70. Preferably, the surface of the shelf is provided with anti-slip texture 74. More preferably, the outer surface of the top of the shell is the shelf.

[0139] (4) The shell is provided with any one or more of the following: an air inlet channel, an exhaust channel, a feed channel, and a discharge channel. Specifically, an air inlet channel (not shown in the figure) is provided to allow external gas to enter the internal space 11 of the reaction chamber body 10, for example, by connecting the air inlet channel and the air inlet through the aforementioned air inlet pipe; an exhaust channel is provided to allow the gas in the internal space of the reaction chamber body 10 to be discharged, for example, by connecting the exhaust port and the exhaust channel through the aforementioned exhaust pipe; a feed channel 11a is provided to allow the synthesis reaction liquid to enter the internal space 11 of the reaction chamber body 10 for in vitro biosynthesis (e.g., Figure 12 As shown); the reaction products are discharged through the discharge channel 11b (e.g. Figure 12 ).

[0140] Example 4

[0141] In this embodiment, the following in vitro transcription-translation system was used to test the expression of the target protein EGFP:

[0142] (1) First type of experimental setup: as in Example 1 Figure 1 The reaction apparatus has a first protrusion arranged on the inner wall of the reaction chamber body and a second protrusion arranged on the outer surface of the partition.

[0143] (2) The second type of experimental setup: The difference between the second and third types of experimental setups is that the inner wall of the reaction chamber and the outer surface of the separator are both smooth;

[0144] (3) The third type of experimental setup: 24-well plate.

[0145] 1 L of the same IVTT reaction solution (in vitro transcription and translation system) was added to the internal space of the reaction chamber of the first and second experimental devices, and the tests were conducted under the same rotation speed and temperature conditions.

[0146] Add 300 μL of the same IVTT reaction solution to a 24-well plate and react at the same temperature as a control.

[0147] The EGFP fluorescence values ​​of samples were measured after reactions at 1h, 2h, 3h, 4h, 5h, and 6h, respectively. The results are as follows. Figure 13 As shown in the figure, the improved reaction device of the present invention greatly increases the expression yield of the target protein, and the reaction device with protrusions has a higher fluorescence value of the IVTT product, that is, better IVTT activity.

[0148] Samples were taken at different time points after the reaction to test the EGFP fluorescence value. It was found that the IVTT product had a higher fluorescence value in the reactor with a raised inner wall, which means that the IVTT activity was better.

[0149] It should be noted that in the above embodiments, air intake and material feeding can be accomplished by pumping, and exhaust and material discharge can be accomplished by suction.

Claims

1. A reaction apparatus for in vitro cell-free biosynthesis, characterized in that, include: The reaction chamber body is used to contain the synthesis reaction solution for in vitro biosynthesis. The reaction chamber body is provided with an opening, which has an inlet for allowing the synthesis reaction solution to enter the reaction chamber body and an outlet for discharging the reaction product. A partition, which is used to partially divide the internal space of the reaction chamber body. The partition includes at least one partition element, which is fixedly disposed on the inner wall of the reaction chamber body. The separator is sheet-shaped. At least one of the partition members has its left, right, and lower edges in contact with the inner wall of the reaction chamber body, while its upper edge does not contact the inner wall of the reaction chamber body, thus forming an incomplete partition, so that the partitioned spatial regions are interconnected. Each of the aforementioned separators is independently configured; and A rotating part, which is used to rotate the reaction chamber body.

2. The reaction apparatus according to claim 1, characterized in that: in, The reaction chamber body is a tank.

3. The reaction apparatus according to claim 2, characterized in that: in, The reaction chamber body is a horizontal tank.

4. The reaction apparatus according to claim 1, characterized in that: in, The multiple separators are evenly spaced.

5. The reaction apparatus according to any one of claims 1-4, characterized in that: in, The length of the separator extends in a curved or broken line, and / or the line connecting the two ends of the length of the separator forms an angle α between the line and the central axis of the internal space of the reaction chamber body, which is in the same direction as the length extension direction.

6. The reaction apparatus according to claim 5, characterized in that: in, The included angle α is less than or equal to 60°.

7. The reaction apparatus according to claim 5, characterized in that: in, The included angle α is less than or equal to 45°.

8. The reaction apparatus according to claim 5, characterized in that: in, The included angle α is less than or equal to 20°.

9. The reaction apparatus according to any one of claims 1-4, characterized in that: in, One side of the sheet-like separator faces the inner wall of the reaction chamber body, and / or a recessed area is provided on the other side of the sheet-like separator facing away from the inner wall.

10. The reaction apparatus according to claim 9, characterized in that: in, When one side of the sheet-like separator faces the inner wall of the reaction chamber body, the angle between the sheet-like surface of the separator and the opposite inner wall is not equal to 90°.

11. The reaction apparatus according to any one of claims 1-4, characterized in that: in, In use, the reaction is carried out by rotating the reaction chamber body.

12. The reaction apparatus according to claim 11, characterized in that: in, When the reaction chamber body rotates to carry out the reaction, the horizontal central axis of the reaction chamber body is deviated from the horizontal state.

13. The reaction apparatus according to any one of claims 1-4, characterized in that: in, The separator is provided with at least one flow hole.

14. The reaction apparatus according to any one of claims 1-4, characterized in that: in, When the partition is disposed on the inner wall, there is at least one discontinuity between the partition and the inner wall.

15. The reaction apparatus according to claim 13, characterized in that: in, The flow holes on at least one of the separators are uniformly distributed.

16. The reaction apparatus according to claim 14, characterized in that: in, At least one of the partitions is equidistant from the discontinuities.

17. The reaction apparatus according to any one of claims 1-4, characterized in that: in, The opening also has a ventilation unit, which has an air inlet and / or an exhaust outlet. The air inlet is used to allow external gas to enter the internal space of the reaction chamber body, and the exhaust outlet is used to allow gas to exit the internal space.

18. The reaction apparatus according to claim 17, characterized in that: in, One or more of the feed inlet, the discharge outlet, the air inlet, and the exhaust outlet are the same opening.

19. The reaction apparatus according to claim 17, characterized in that, Also includes: An intake unit and / or an exhaust unit, wherein the intake port allows outside gas to enter the internal space through the intake unit, and the exhaust port allows gas from the internal space to exit through the exhaust unit.

20. The reaction apparatus according to claim 19, characterized in that: in, The air intake unit includes at least one air intake pipe, one end of which faces the air intake port and supplies the outside gas to the internal space.

21. The reaction apparatus according to claim 19, characterized in that: in, The air intake unit includes at least one air intake pipe, one end of which extends from the air intake port into the internal space.

22. The reaction apparatus according to claim 21, characterized in that: in, At least one of the air intake pipes extends horizontally along the body of the reaction chamber.

23. The reaction apparatus according to claim 21, characterized in that: in, At least one air outlet is provided on the wall of at least one portion of the air intake pipe that enters the internal space.

24. The reaction apparatus according to claim 23, characterized in that: in, At least one of the air outlets is evenly distributed, and / or one end of the air intake pipe entering the internal space is sealed.

25. The reaction apparatus according to claim 19, characterized in that: in, The exhaust unit includes an exhaust pipe.

26. The reaction apparatus according to claim 20, characterized in that: in, The exhaust unit includes an exhaust pipe, and a portion of the intake pipe is disposed in the exhaust pipe between its two ends. One end of the intake pipe extends out of the exhaust pipe and into the internal space, while the other end of the intake pipe extends out of the exhaust pipe and is connected to an air source.

27. The reaction apparatus according to any one of claims 1-4, characterized in that, Also includes: A cover assembly having a cover adapted to the feed inlet for covering the feed inlet.

28. The reaction apparatus according to claim 27, characterized in that: in, The lid assembly also includes a sealing ring, a lid latch, and a locking handle.

29. The reaction apparatus according to claim 25, characterized in that: Also includes: A cover assembly having a cover adapted to the feed inlet for covering the feed inlet.

30. The reaction apparatus according to claim 29, characterized in that: in, The exhaust unit also includes an exhaust port, which is disposed on the cover, and the exhaust pipe is connected to the internal space through the exhaust port.

31. The reaction apparatus according to claim 1, characterized in that, in, The rotating part rotates the reaction chamber body by being rotatably connected to the reaction chamber body.

32. The reaction apparatus according to claim 31, characterized in that, in, The rotating part is also provided with a rotating wheel, the reaction chamber body is disposed on the rotating wheel, and the rotating wheel rotates as the reaction chamber body rotates.

33. The reaction apparatus according to claim 1, characterized in that: in, The rotating part includes a driving unit, which drives the reaction chamber body to rotate.

34. The reaction apparatus according to any one of claims 1-4, characterized in that, Also includes: A temperature control device is used to maintain the reaction at a predetermined temperature.

35. The reaction apparatus according to claim 34, characterized in that, in, The temperature control device is used to regulate the temperature of the gas entering the reaction chamber body in order to maintain the predetermined temperature conditions.

36. The reaction apparatus according to any one of claims 1-4, characterized in that: in, At least one first protrusion is arranged on the inner wall of the reaction chamber body.

37. The reaction apparatus according to any one of claims 1-4, characterized in that: in, At least one second protrusion is arranged on the outer surface of the separator.

38. The reaction apparatus according to any one of claims 1-4, characterized in that, Also includes: A housing adapted to the reaction chamber body for accommodating the reaction body.

39. The reaction apparatus according to claim 38, characterized in that: in, The housing has any combination of one or more of the following features: The housing is provided with an opening and closing door; A display screen is provided on the outside of the housing; The outer side of the housing is provided with a shelf for placing items; The housing is provided with any one or more of the following: an air inlet channel for allowing external gas to enter the internal space of the reaction chamber body; an exhaust channel for allowing gas to exit the internal space of the reaction chamber body; a feed channel for allowing the synthesis reaction liquid to enter the internal space of the reaction chamber body for in vitro biosynthesis; and an outlet channel for allowing the reaction products to exit.

40. The reaction apparatus according to claim 39, characterized in that: in, The opening and closing door is provided with a ventilation hole; The display screen is located on the outer surface of the top of the housing; The surface of the shelf is provided with anti-slip texture.

41. The reaction apparatus according to claim 39, characterized in that: in, The outer surface of the top of the housing is the shelf.

42. An in vitro biosynthesis method, characterized in that: A reaction using the reaction apparatus according to any one of claims 1-41 to contain a reaction solution for synthesis for in vitro biosynthesis.

43. The in vitro biosynthesis method according to claim 42, characterized in that: in, The volume of the reaction solution used for the synthesis of the reaction is less than or equal to 30% of the volume of the reaction chamber body.

44. The in vitro biosynthesis method according to claim 42 or 43, characterized in that: in, During the reaction process, external gas is introduced into the internal space, and / or gas is discharged from the internal space.

45. The in vitro biosynthesis method according to any one of claims 42-43, characterized in that: in, During the reaction process, the reaction chamber body of the reaction device is rotated.

46. ​​The in vitro biosynthesis method according to claim 45, characterized in that: in, The horizontal central axis of the reaction chamber body is deviated from the horizontal state.

47. The in vitro biosynthesis method according to claim 45, characterized in that: in, The rotational speed is less than or equal to 120 r / min.

48. The in vitro biosynthesis method according to claim 44, characterized in that: in, During the reaction process, the reaction chamber body of the reaction device is rotated.

49. The in vitro biosynthesis method according to claim 48, characterized in that: in, The horizontal central axis of the reaction chamber body is deviated from the horizontal state.

50. The in vitro biosynthesis method according to claim 48, characterized in that: in, The rotational speed is less than or equal to 120 r / min.

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