Reaction piece, reaction vessel, reaction system and cell-free reaction method

Abstract

The application relates to the technical field of cell-free reaction, and discloses a reaction piece, a reaction container, a reaction system and a cell-free reaction method, which solve the technical problem of how to make the cell-free reaction more sufficient. The reaction piece comprises a main body section and a bending section, the bending section is connected to the end of the main body section, the included angle between the bending section and the main body section is theta 1, [0 DEG < theta 1 <= 180 DEG], the width of the bending section is K1, the width of the main body section is K2, K1:K2=1:3, the length of the reaction piece is L1, the height of the reaction container is L2, L1:L2<90%, the reaction piece is installed in the length direction and perpendicular to the inner wall of the container main body of the reaction container, and is installed in the width direction and perpendicular to the top wall of the container base of the reaction container. According to the above technical scheme, the reaction piece is used to make the reaction liquid in the reaction container more sufficient.

Description

Technical Field

[0001] This invention relates to the field of cell-free reaction technology, and more specifically, to reaction components, reaction containers, reaction systems, and cell-free reaction methods. Background Technology

[0002] Cell-free reaction is a broad concept, generally referring to a series of biochemical reactions that do not rely on intact living cells. In vitro cell-free protein synthesis is a specific form of cell-free reaction, referring to a biochemical reaction that uses exogenous mRNA or DNA as a template to synthesize proteins under in vitro conditions using enzymes, amino acid substrates, and energy. In the last 20 years, extensive research has been conducted on the reaction mechanisms and regulation, energy supply, genetic template stability, and reaction tool design and operation in in vitro cell-free protein synthesis systems.

[0003] Cell-free reactions require a reaction vessel, which is a biochemical experimental apparatus. When conducting a cell-free reaction, a reaction solution is added to the reaction vessel (for example, in an in vitro cell-free protein synthesis reaction, a reaction solution is added to the reaction vessel). The degree of reaction sufficiency of the reaction solution in the reaction vessel is crucial to the quality of the reaction and will directly affect parameters such as protein synthesis yield.

[0004] Therefore, how to ensure a high degree of complete reaction in the reaction vessel, that is, to enable the reaction solution to react more fully, is a major technical problem that needs to be solved in the field of in vitro cell-free reaction technology. Summary of the Invention

[0005] The present invention aims to disclose and protect a reaction element, a reaction vessel, a reaction system, and a cell-free reaction method to solve the technical problem of how to enable the reaction solution to react more fully as mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A reaction element includes a main body segment and a bent segment, the bent segment being connected to the end of the main body segment, and the included angle between the bent segment and the main body segment being θ1, [0° < θ1 ≦ 180°].

[0008] Furthermore, the connection between the bent section and the main body section is arc-shaped.

[0009] Furthermore: the width of the bending segment is K1, and the width of the main body segment is K2, K1:K2 = 1:3. Preferably, the reaction element has an inclined draft angle θ2 in both the width direction and the length direction, [0.5°≦θ2≦0.8°].

[0010] Furthermore: the main body segment and the bending segment are integrally formed; or the main body segment and the bending segment are separately formed, and the bending segment is detachably fixed to the end of the main body segment.

[0011] Furthermore: the main body segment is configured as a straight line;

[0012] The bending section is configured as a straight line, or

[0013] The bending segment is configured as an arc shape, and the concave direction of the arc-shaped bending segment is concave in the direction of its bending.

[0014] A reaction vessel includes a container body having an inner cavity for adaptably installing the aforementioned reaction components. After the reaction components are installed in the inner cavity, multiple partially sealed partitioned areas are formed.

[0015] Furthermore: The compatibility installation between the reaction vessel and the reaction components shall be adapted using one or more of the following installation ratios:

[0016] (1) The length of the reaction element is L1, the height of the reaction vessel is L2, and L1:L2<90%;

[0017] (2) The total straight width of the reaction vessel from the main body section to the bending section is L3, and the diameter of the reaction vessel is L4, where L3:L4<45%.

[0018] A cell-free reaction system includes the aforementioned reaction element and the aforementioned reaction container. The reaction element is installed perpendicular to the inner wall of the container body in the length direction and perpendicular to the bottom surface of the inner cavity of the container body in the width direction.

[0019] Furthermore: the reaction element is fixed in the reaction vessel using any of the following fixing methods:

[0020] (1) The reaction element is vertically fixed to the inner wall of the container body in the length direction;

[0021] (2) The reaction element is vertically fixed to the bottom surface of the inner cavity of the container body in the width direction;

[0022] (3) The reaction element is vertically fixed to the inner wall of the container body in the length direction and vertically fixed to the bottom surface of the container body in the width direction.

[0023] Preferably, the reaction element is provided with rounded corners between the inner wall of the container body and / or the top wall of the container base;

[0024] Preferably, the number of the reactants is one or more;

[0025] More preferably, the number of the reaction elements is set to 1 to 8.

[0026] A cell-free reaction method, using the aforementioned cell-free reaction system to perform a cell-free reaction.

[0027] Furthermore, the reaction method includes the following steps:

[0028] Add reaction materials into the reaction vessel;

[0029] The reaction vessel is placed at a preset tilt angle θ3;

[0030] The driving reaction vessel rotates at a preset speed r, and the rotation process lasts for a preset time T;

[0031] Preferably, the length of the reaction vessel is L1, and the height of the reaction material in the reaction vessel is L5, where L1:L5>200%.

[0032] In summary, the present invention has the following beneficial effects: Through the obstruction of the reaction components, the reaction liquid achieves collision stirring and spreading with the reaction components as it rotates with the container body. Simultaneously, the bending section set relative to the main body section allows the reaction liquid to alternately flow from different partitioned areas formed between the reaction components. When flowing outward from one partitioned area to another, the surface of the bending section increases the probability of the reaction liquid being impacted and obstructed. Furthermore, due to the rotation of the reaction container, the reaction liquid within the partitioned area easily forms vortices (eddies) under the combined effect of rotation and the obstruction of the bending section. This allows for better agitation of the synthetic raw materials within a single partitioned area, increasing the probability of mixing the various parts of the raw materials within the partitioned area during the reaction. In other words, the bending section enhances the mixing efficiency of the raw materials within the partitioned area, ensuring better mixing of the raw materials in each partitioned area. Overall, it prevents the reaction liquid from accumulating in corners, thereby improving the efficiency of the cell-free reaction. At the same time, the bending section also increases the area of ​​the reaction liquid that is spread, further improving the efficiency of the cell-free reaction. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the three-dimensional structure of a cell-free reaction system;

[0035] Figure 2 This is a top view of the reaction vessel;

[0036] Figure 3 This is a front view of the reaction vessel;

[0037] Figure 4 This is a frontal cross-sectional view of the cell-free reaction system;

[0038] Figure 5 This is a top view of the cell-free reaction system;

[0039] Figure 6 This is a schematic diagram of the cell-free reaction method.

[0040] Figure 7 A schematic diagram of a structure in which the number of reactants in the reaction vessel is set to 0.

[0041] Figure 8 A schematic diagram showing a reaction vessel containing only one reactant.

[0042] Figure 9 A schematic diagram showing a reaction vessel containing two reactants.

[0043] Figure 10 A schematic diagram showing a reaction vessel containing three reactants.

[0044] Figure 11 A schematic diagram showing a reaction vessel containing 5 reactants.

[0045] Figure 12 A schematic diagram showing a reaction vessel containing 6 reactants.

[0046] Figure 13 A schematic diagram showing a reaction vessel containing 7 reactants.

[0047] Figure 14 A schematic diagram showing a reaction vessel containing 8 reactants.

[0048] Figure 15 A comparison of RFU test results for in vitro protein synthesis biological reactions using a reaction vessel with four reaction elements and a reaction vessel without reaction elements (smooth inner wall);

[0049] Figure 16 A comparison of RFU test results for in vitro protein synthesis bioreactors using a reaction vessel with four reaction elements and a reaction vessel with a conventional stirring blade.

[0050] Figure reference numerals: 1. Reaction component; 1-1. Main body section; 1-2. Bending section; 2. Container body; 2-1. Cup mouth; 2-2. Flared ear; 2-3. Protrusion; 3. Container base;

[0051] K1, the width of the bending section; K2, the width of the main body section;

[0052] L1, length of the protrusion; L2, height of the reaction vessel; L3, length of the reaction element; L4, diameter of the reaction vessel; L5, height of the reaction liquid in the reaction vessel. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] A reaction device, used in the field of cell-free reactions, is mainly used to increase the probability of the reaction solution being agitated and spread during rotation, shaking, and swirling in a cell-free reaction vessel, thereby improving the degree of reaction completion. For example... Figure 1 As shown, the reaction component 1 includes a main body segment 1-1 and a bent segment 1-2, which are integrally formed. The bent segment 1-2 is connected to the end of the main body segment 1-1. Figure 1 and Figure 2 As shown, the included angle between the bent segment 1-2 and the main body segment 1-1 is θ1, [0° < θ1 ≦ 180°], and the connection between the bent segment 1-2 and the main body segment 1-1 is arc-shaped; in other embodiments, the main body segment 1-1 and the bent segment 1-2 can also be separately arranged, for example, the bent segment 1-2 can be detachably fixed to the end of the main body segment 1-1. The side with the included angle θ1 between the bent segment 1-2 and the main body segment 1-1 serves as the side of the stirring reaction liquid.

[0055] It should be noted that the included angle θ1 between the bent section 1-2 and the main body section 1-1 is preferably 135°-140°, and more preferably 135°. The design principle of setting the reaction element 1 of the present invention as a bent approximately L-shaped structure is that when the reaction element 1 is designed as an approximately L-shaped structure, the main body section 1-1 of the reaction element 1 can play a better stirring role (when the reaction container rotates, the reaction liquid impacts the main body section under the action of gravity), so as to increase the mixing effect of the reaction liquid in the reaction container, thereby improving the degree of reaction of the reaction liquid; at the same time, when the reaction element 1 is installed in the reaction container (see the following description of the specific installation method of the reaction element 1 in the reaction container), and when the number of reaction elements 1 is set to multiple, the main body section 1-1 can play a certain partitioning role, that is, to divide the internal space of the reaction container into partially enclosed partitions, providing space for the reaction liquid to react in different partition areas, but not completely restricting the reaction to a certain partition area. At this time, the reaction liquid will react alternately in different partition areas. Therefore, it can be seen that the partitioning function of the main section 1-1 is that, while playing a stirring role, it also provides partitioned areas so that the reaction liquid can also react in each partitioned area in a regular alternation.

[0056] The incompletely closed partition refers to, in combination with Figure 8 As shown, when the number of reactants 1 inside the container body is set to 1, the incompletely sealed partition refers to a gap between the end of reactant 1 and the inner wall of the container body; that is, the end of reactant 1 is not sealed due to contact with the inner wall of the container body at any position. (Combined with...) Figures 9-14 and combination Figure 5 As shown, the number of reaction elements 1 inside the container body is set to 2, 3, 4, 5, 6, 7 or 8. In this case, the incompletely closed partition means that the end of any reaction element 1 is a free end, that is, the end of any reaction element 1 is not sealed by contact with any other reaction element 1, nor is it sealed by contact with the inner wall of any position of the container body.

[0057] The stirring action includes active stirring and passive stirring. Passive stirring refers to the reaction component being fixed to the inner wall of the container body. The movement of the reaction component must be synchronized with the rotation of the container body. During the rotation of the container body, the reaction liquid inside can be mixed. In this process, the mixed reaction liquid will be resisted by the reaction component when it passes through the reaction component that is stationary relative to the container body. Thus, the reaction component is used to disturb the reaction liquid, that is, to provide stirring force to the reaction liquid, and stirring is carried out. Secondly, as mentioned above, during the rotation of each reactant along with the container body, they will inevitably encounter the reaction liquid being stirred inside. When the reaction liquid encounters the rotating reactant, the rotating reactant will generate a certain resistance to the reaction liquid. This resistance will cause the reaction liquid in contact with the reactant to adhere to the surface of the reactant for a shorter time compared to the uncontacted part. Furthermore, due to the presence of each reactant, the surface area that the reaction liquid can contact during the rotation of the container body is increased. As the reaction liquid flows through the reactant, it will adhere to and spread onto each reactant due to the resistance. Therefore, compared to no reactant, the design of multiple reactants in this application can increase the area of ​​the reaction liquid adhering to the solid surface inside the container body at a certain point in time, thereby achieving temporary thinning overall.

[0058] Active stirring refers to a process where the reactant is not fixed to the inner wall of the container body, and regardless of whether the container body rotates, the reactant is self-driven by an external rotating shaft. Under the driving force of the external rotating shaft, the reactant actively stirs relative to the container body (see the example below for a certain installation method of the rotating shaft). After active stirring, the reaction liquid inside can also be mixed. In this process, the mixed reaction liquid is disturbed by the moving reactant, which provides a stirring force to the reaction liquid. Secondly, as mentioned above, during the disturbance process, each reactant will inevitably encounter the reaction liquid being stirred inside it. When the reaction liquid encounters the rotating reactant, the rotating reactant will generate a certain resistance to the reaction liquid. This resistance will cause the reaction liquid in contact with the reactant to adhere to the surface of the reactant for a shorter time compared to the uncontacted part. Furthermore, due to the presence of each reactant, the surface area that the reaction liquid can contact during the rotation of the container body is increased. As the reaction liquid flows through the reactant, it will adhere to and spread onto each reactant due to the resistance. Therefore, compared to no reactant, the design of multiple reactants in this application can increase the area of ​​the reaction liquid adhering to the solid surface inside the container body at a certain point in time, and also achieve temporary thinning overall.

[0059] The aforementioned partitioning effect refers to the fact that by dividing the inner cavity of the container into partitions, the reaction liquid can react regularly and alternately in the individual areas of each partition. This regular and alternating reaction in different areas may increase the probability of reaction liquids that were originally in different positions randomly stopping in a certain partition area and coming into contact with each other, thereby improving the mixing degree and reaction degree of the reaction liquid.

[0060] When the reactor 1 is designed with an approximately L-shaped structure, the bending sections 1-2 of the reactor 1 can effectively generate vortices. The principle is that as the reaction liquid flows alternately from different partitioned areas formed between the reactor components, the bending section surface increases the probability of the reaction liquid being impacted and blocked when flowing outward from one partitioned area to another. Furthermore, due to the rotation of the reaction vessel, the reaction liquid within the partitioned area easily forms vortices (eddies) under the combined effect of rotation and the blocking effect of the bending section. This allows for better movement of the synthetic raw materials within a single partitioned area, increasing the probability of mixing the raw materials in each partitioned area during the reaction. In other words, the bending section enhances the mixing efficiency of the raw materials within the partitioned area, ensuring better mixing of the raw materials in each partitioned area. Overall, it prevents the reaction liquid from accumulating in corners, thereby improving the efficiency of the cell-free reaction. Simultaneously, the bending section also increases the area over which the reaction liquid is spread, further improving the efficiency of the cell-free reaction.

[0061] Regarding the setting of the included angle θ1 in this invention, since if the angle value of included angle θ1 is too large, it is no different from a straight structure, and if the angle is too small, it is easy to form liquid accumulation (aggregation of synthetic raw materials) at the included angle position. Therefore, in this embodiment, the included angle θ1 is preferably 135°.

[0062] like Figure 1 As shown, in this embodiment, the main body segment 1-1 is configured as a straight line, and the bending segment 1-2 is configured as a straight line. Alternatively, in another embodiment, the main body segment 1-1 is configured as a straight line, while the bending segment 1-2 is configured as an arc, wherein the concave direction of the arc-shaped bending segment is concave in the direction of its bending.

[0063] It should be noted that the arc-shaped connection between the bending segment 1-2 and the main body segment 1-1 means that the connection between the bending segment and the main body segment 1-1 is an arc-shaped segment or a rounded corner. When the connection between the bending segment 1-2 and the main body segment 1-1 is an arc-shaped segment, the reaction component 1 includes three parts: the main body segment 1-1, the arc-shaped segment, and the bending segment 1-2. When the connection between the bending segment 1-2 and the main body segment 1-1 is a rounded corner, the reaction component 1 includes two parts: the main body segment 1-1 and the bending segment 1-2, and the connection between the main body segment 1-1 and the bending segment 1-2 is machined with a rounded corner.

[0064] It should be noted that the reaction element 1 has an approximately L-shaped structure, preferably an L-shaped plate structure, that is, the reaction element 1 is a reaction partition.

[0065] like Figure 2 As shown, the width of the bending segment 1-2 is K1, and the width of the main segment 1-1 is K2. Preferably, K1:K2 = 1:3. Of course, the ratio of K1:K2 can also be other ratios. The principle behind setting the K1:K2 ratio is that K2 is usually greater than K1. Since K2 represents the width of the main segment 1-1 and K1 represents the width of the bending segment 1-2, if the width of the main segment 1-1 is smaller and the width of the bending segment 1-2 is larger, it is easier to seal the partition between adjacent main segments 1-1, preventing the reaction liquid from reacting alternately in adjacent partition areas, thus affecting the reaction quality. Therefore, K2 is usually set to be greater than K1.

[0066] In addition, such as Figure 2 and Figure 3 As shown, the reaction vessel 1 of the present invention has an inclined draft angle θ2 in both the width and length directions, [0.5°≦θ2≦0.8°], and is generally spindle-shaped. This inclined draft angle θ2 refers to the angle that tapers towards the end along the width direction and the angle that tapers upwards along the length direction. The beneficial effect of this structural design is that when the reaction vessel is tilted, the reaction vessel 1 is also tilted. When the reaction vessel rotates, the reaction liquid inside the reaction vessel flows more easily along the draft angle of the reaction vessel 1, promoting the flow of the reaction liquid and increasing the flow effect.

[0067] A reaction vessel for use in cell-free reactions. (Combined with...) Figure 1 , Figure 4 , Figure 7 As shown, the reaction vessel includes a container body 2 and a container base 3. The vertical cross-section of the container body 2 is trapezoidal, and the top width of the container body 2 is greater than the bottom width, which is an open design. In this invention, the principle behind this structural design of the container body 2 is that when the reaction vessel is placed at an angle, the container body 2 is also in an angled state. At this time, during the biochemical reaction process when the reaction vessel rotates, the reaction liquid inside the container body 2 flows more easily along the trapezoidal inclined side of the container body 2, promoting the flow of the reaction liquid, increasing the flow effect, and improving the sufficiency of the reaction.

[0068] The following content describes the design of the main structure of container 2.

[0069] The inner wall of container body 2 has a draft angle of 0.8°-1° that tapers from bottom to top. This means the wall thickness of container body 2 is a bottom-thicker-top structural design, which increases the structural strength of the bottom of the reaction vessel, as the bottom is used for load-bearing. Alternatively, both the inner and outer walls of container body 2 can have a draft angle of 0.4°-1° that tapers from bottom to top, also achieving a bottom-thicker-top structural effect. This design similarly increases the structural strength of the bottom of the reaction vessel.

[0070] The container body 2 has a cup opening 2-1 at its top. The cup opening 2-1 is an open opening that serves as a feed inlet, discharge outlet, air inlet, and air outlet. The feed inlet, discharge outlet, air inlet, and air outlet are integrated into the cup opening 2-1. In other embodiments, one or more of the feed inlet, discharge outlet, air inlet, and air outlet can be designed as separate structures. In this invention, a flanged lug 2-2 is provided near the cup opening 2-1 on the container body. The flanged lug 2-2 is integrally formed with the container body 2. The main function of the flanged lug 2-2 is to facilitate manual handling of the reaction container by the experimental operator.

[0071] In order to improve the ability of the container body 2 to resist bending deformation, the container body 2 of the present invention has an indentation from the outside to the inside on the outer wall and a corresponding protrusion 2-3 formed on the inner wall. The protrusion 2-3 is similar to the structural design of a reinforcing rib, thereby improving the ability of the container body 2 to resist bending deformation.

[0072] Typically, the protrusions 2-3 are positioned along the height of the container body 2. For example, in one embodiment, the container body 2 is a polygonal structure with multiple corners. The outer wall of the container body 2 is concave from the outside to the inside at the corners, and the inner wall of the container body forms corresponding protrusions 2-3 at the corners. Furthermore, in one embodiment, the cross-sectional shape of the protrusions 2-3 is rectangular, with the bottom end of the protrusions 2-3 flush with the container base 3, and the top end of the protrusions 2-3 lower than the top end of the container body 2. The length of the protrusions 2-3 is L1, and the height of the reaction container is L2, with L1:L2 < 90%. Simultaneously, the connection points between the protrusions 2-3 and the container body 2 and container base 3 are rounded to avoid forming right-angle gaps on the inner wall of the container body 2, which would facilitate the residue of the reaction liquid.

[0073] In addition to its own structural features, the reaction vessel of the present invention is mainly used for the installation of the aforementioned reaction element 1 within its internal cavity. Therefore, from the perspective of the compatible installation between the reaction vessel and the reaction element 1, some structural design features need to be explained.

[0074] The compatibility installation between the reaction vessel and reaction element 1 shall be adapted using any one or more of the following installation ratios:

[0075] (1) The length of reaction vessel 1 is L3, the height of reaction vessel is L2, and L3:L2<90%.

[0076] The principle behind setting the L3:L2 ratio is that L2 represents the height of the reaction vessel, and L3 represents the length (vertical height) of reaction element 1. As explained earlier, reaction element 1 mainly serves to stir, partition, and create vortices. In reality, when the reaction vessel is used for a reaction, the volume of the reaction liquid added inside is usually around 10%-35% of the total volume of the reaction vessel. Therefore, the height of the reaction liquid inside the reaction vessel is usually also around 10%-35% of the total height. Theoretically, the ratio of the length L3 of reaction element 1 to the height L2 of the reaction vessel should be set to 10%-35%. Even if the reaction vessel is tilted during the reaction (usually at a vertical tilt angle of around 30°), it is not necessary to set the ratio of the length L3 of reaction element 1 to the height L2 of the reaction vessel to be close to 90%. Why is it necessary to set it to be close to but less than 90%? The reason is that, since the reaction vessel is an open design, the reaction liquid may splash when it hits the bottom reaction element during the reaction. Therefore, the larger the ratio of the length L3 of the reaction element 1 to the height L2 of the reaction vessel, the closer the length L3 of the reaction element 1 is to the height L2 of the reaction vessel. In this case, the top reaction element 1 can play a certain role in preventing splashing.

[0077] (2) Combination Figure 2 and Figure 5 As shown, the total straight-line width of the reaction element 1 from the main body section 1-1 to the bending section 1-2 is K1+K2, and the diameter of the reaction vessel is L4, where (K1+K2):L4<45%, preferably 35%. When the proportion is greater than or equal to 50%, the ends of the two opposite reaction elements 1 will abut against each other, forming a completely sealed space, and this situation must be excluded; when the proportion is less than 50%, the ends of the two opposite reaction elements 1 will not abut against each other, forming an incompletely divided area, and this situation is applicable to the technical solution of the present invention.

[0078] It should be noted that the primary premise of the size design of K1+K2 is to avoid forming a completely closed partition. The specific size of K1+K2 and its proportional relationship with the diameter L4 of the reaction vessel can be selected according to the actual situation.

[0079] It should also be noted that the diameter L4 of the reaction vessel should be understood as the width of the reaction vessel along the straight line of the total straight width K1+K2. When the reaction vessel is circular, the diameter L4 is the diameter of the circular vessel; when the reaction vessel is of other shapes, the diameter L4 is the distance between the edge of the reaction vessel used to mount the reaction component 1 and the edge of the reaction vessel located in the direction of the width extension of the reaction component 1.

[0080] (3) Figure 4 As shown, the length of reaction vessel 1 is L1, and the height of the reaction liquid in the reaction vessel is L5, where L1:L5>200%.

[0081] As mentioned above, when a reaction vessel is used for a reaction, the volume of the reaction liquid added inside the vessel typically accounts for about 10%-35% of the vessel's volume. Therefore, the height of the reaction liquid inside the vessel is also approximately 10%-35% of its total height. To ensure that reaction component 1 achieves its intended function, the length L1 of reaction component 1 must typically be at least twice the height of the reaction liquid within the reaction vessel, i.e., L1:L5 > 200%.

[0082] Based on the foregoing, the reaction vessel is adapted to install the reaction component 1 to form a cell-free reaction system. This cell-free reaction system includes the aforementioned reaction component 1 and the aforementioned reaction vessel. In this embodiment, the cell-free reaction system uses a disposable reaction cup as an example. Figure 1 As shown, the cup body of this disposable reaction cup is equivalent to the main body of the container, and the paddle installed inside the cup body is equivalent to the reaction element.

[0083] In this cell-free reaction system (disposable reaction cup), the reaction element 1 is installed perpendicular to the inner wall of the container body 2 in the length direction and perpendicular to the top wall of the container base 3 in the width direction. Furthermore, the reaction element 1 is fixed in the reaction container using any of the following fixing methods:

[0084] (1) The reaction element 1 is vertically fixed to the inner wall of the container body 2 in the length direction;

[0085] (2) The reaction vessel 1 is vertically fixed to the top wall of the container base 3 in the width direction;

[0086] (3) The reaction element 1 is vertically fixed to the inner wall of the container body 2 in the length direction, and at the same time, it is vertically fixed to the top wall of the container base 3 in the width direction.

[0087] Preferably, the reaction element 1 is provided with rounded corners between the inner wall of the container body 2 and / or the top wall of the container base 3.

[0088] Preferably, the number of reactants 1 is one or more, and the number of reactants 1 is set to 1-8, as shown below. Figure 8 , Figure 9 , Figure 10 , Figure 5 , Figure 11 , Figure 12 , Figure 13 , Figure 14 As shown.

[0089] Alternatively, the reactant 1 can be rotatably installed inside the container body 2. When the reactant 1 is rotatably installed inside the reaction vessel, a rotating shaft is required to connect all the reactants 1 into a whole, and the rotation of the shaft drives the reactants 1 to rotate synchronously. For example, the reactant 1 can be slidably installed on the inner wall of the container body 2, or the reactant 1 can be separated from the inner wall of the container body 2 with a gap.

[0090] The rotating shaft is driven by an external drive component, so that the rotating shaft can start to rotate under the action of the external drive, thereby driving the components of the reaction component 1 to rotate synchronously.

[0091] When the rotating shaft is connected to the reaction component 1, multiple connecting rods are set up. One end of each connecting rod is connected to the end of the reaction component 1 near the inner wall of the container body 2 (one connecting rod corresponds to one reaction component 1), and the other end of each connecting rod is fixed to the rotating shaft at the center position to realize the component of jointly driving the reaction component 1.

[0092] A cell-free reaction method, using the aforementioned cell-free reaction system to perform a cell-free reaction, taking a cell-free protein synthesis reaction method as an example, such as... Figure 6 As shown, the reaction method includes the following steps:

[0093] A100. Add reaction materials to the reaction vessel.

[0094] The reaction raw materials include templates, amino acids, energy substances, cell lysis fluid, cofactors, and nucleotides, etc., which are also known as reaction solutions.

[0095] A200. Place the reaction vessel at a preset tilt angle θ3.

[0096] The reaction vessel is placed inside the cup holder, which is tilted at 30°, so that the reaction vessel is tilted at 30° simultaneously.

[0097] The 30° inclination refers to the angle between the inclination and the vertical line being 30°.

[0098] A300 drives the reaction vessel to rotate at a preset speed r for a preset time T.

[0099] An external motor drives the cup holder to rotate at a preset speed r, which is 3-6 r / min, for a preset time T, which is 1-6 hours. During the rotation of the reaction vessel, the reaction liquid is mixed within the vessel through rotation, swishing, and shaking.

[0100] 1. The reason for the rotation is that the external motor drives the cup body to rotate, which in turn drives the reaction vessel to rotate. As a result, the reaction liquid rotates relative to the reaction vessel and impacts the reaction components under the action of gravity, forming eddies and turbulence.

[0101] 2. The reason for the swaying motion is that, due to the centrifugal force of rotation, the reaction liquid sways away from the center of the circle.

[0102] 3. The reason for the shaking is that there is a gap between the reaction vessel and the cup holder. Therefore, when the cup holder rotates, it shakes with the reaction vessel, which in turn causes the internal reaction liquid to shake.

[0103] To verify whether the presence or absence of reaction component 1 affects the effectiveness of in vitro biological experiments on the reaction vessel. For example... Figure 15 The figure shown is a comparison of the RFU test results of in vitro protein synthesis biological reactions in a reaction vessel with four reaction elements and a reaction vessel without reaction elements (with a smooth inner wall).

[0104] The reaction vessel with four reactants (experimental group a1) was used in two experiments: experimental group a1-1 and experimental group a1-2. In experimental group a1-1, 100 ml of reaction solution was added to the reaction vessel, and in experimental group a1-2, 200 ml of reaction solution was added. Similarly, the reaction vessel without reactants (control group b1) was used in two experiments: control group b1-1 and control group b1-2. In control group b1-1, 100 ml of reaction solution was added to the reaction vessel, and in control group b1-2, 200 ml of reaction solution was added.

[0105] Note: 100ml of reaction solution occupies approximately 13% of the container volume, and 200ml of reaction solution occupies approximately 26% of the container volume.

[0106] During the experiment, the experimental parameters for each experiment (experimental group a1-1, experimental group a1-2, control group b1-1, and control group b1-2) in experimental group a1 and control group b1 were set to be the same. During the experiment, the preset rotation speed r was 15 r / min, the preset duration of the reaction rotation process T was 3 h, and the reaction temperature was 30℃.

[0107] Three experiments were conducted on each of the four groups (experimental group a1-1, experimental group a1-2, control group b1-1, and control group b1-2). The results at the end of the experiments were as follows:

[0108] RFU1 RFU2 RFU3 RFU(avg) Experimental group a1-1 (4 leaves, 100 mL) 3661 3712 3357 3576.67 Experimental group a1-2 (4 leaves, 200 mL) 2377 2481 2646 2501.33 Control group b1-1 (100ml without leaves) 2589 2729 2734 2684 Control group b1-2 (200ml without leaves) 1831 2052 1991 1958

[0109] To more intuitively observe the experimental results of the four experiments, through... Figure 15 To display the experimental results, such as Figure 15 The chart shown is a comparison of the RFU test results for the four experiments. It can be seen that:

[0110] When the reaction mixture was 100 ml, experimental group a1-1 was superior to control group b1-1, indicating that the reaction vessel with the reaction element was superior to the reaction vessel without the reaction element. When the reaction mixture was 200 ml, experimental group a1-2 was also superior to control group b1-2, again demonstrating that the reaction vessel with the reaction element was superior to the reaction vessel without the reaction element. Therefore, based on the above experimental results, it can be seen that the reaction vessel with the reaction element in this embodiment has better in vitro biological experimental results than the smooth-walled reaction vessel without the reaction element in this embodiment.

[0111] On the other hand, to verify whether the bending sections 1-2 affect the in vitro biological experimental effects of the reaction vessel, such as... Figure 16 The figure shows a comparison of RFU test results for in vitro protein synthesis bioreactors using a reaction vessel with four reaction elements and a reaction container with six conventional stirring blades. The conventional stirring blades refer to the main body segment 1-1 of reaction element 1 in this embodiment, which does not have a bent section 1-2 at the end of the main body segment 1-1.

[0112] The reaction vessel (experimental group a2) with four reaction components was used in two separate experiments: experimental group a2-1 and experimental group a2-2. 100 ml of reaction solution was added to both the reaction vessel in experimental group a2-1 and experimental group a2-2. A control group (b2) was also used, consisting of a reaction vessel with a conventional stirring blade, in which 100 ml of reaction solution was added.

[0113] During the experiment, the experimental parameters of each experiment (experimental group a2-1, experimental group a2-2, and control group b2) in experimental group 2 and control group 2 were the same. During the experiment, the preset rotation speed r was 15 r / min, the preset duration of the reaction rotation process T was 3 h, and the reaction temperature was 30℃.

[0114] The three experiments (experimental group a2-1, experimental group a2-2, and control group b2) were conducted three times each, and the results at the end of the experiments were as follows:

[0115] RFU Experimental group a2-1 (4 leaves, 100 mL) 3995.33 Experimental group a2-2 (4 leaves, 100 mL) 3467 Control group b2 (6 leaves, 100 mL routine solution) 3252.67

[0116] To more intuitively observe the experimental results of the three experiments, through... Figure 16 To display the experimental results, such as Figure 16 The chart shown is a comparison of the RFU test results for the three experiments. It can be seen that:

[0117] When the reaction material was 100 ml, both experimental groups a2-1 and a2-2 of the four reaction components 1 were better than the control group b2 of the six conventional stirring blades. This indicates that the effect of using reaction component 1 for in vitro biological experiments is better than the effect of using conventional stirring blades for in vitro biological experiments.

[0118] Based on the above experimental results, it can be seen that the reaction vessel with reaction element 1 in this embodiment has better in vitro biological experimental results than the reaction vessel without reaction element 1 in this embodiment (which only has conventional stirring blades).

[0119] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A reaction element, characterized in that, It includes a main body segment and a bending segment, the bending segment being connected to the end of the main body segment, and the included angle between the bending segment and the main body segment being θ1, [0°<θ1≦180°].

2. The reaction element according to claim 1, characterized in that, The connection between the bent section and the main body section is arc-shaped.

3. The reaction element according to claim 1 or 2, characterized in that, The width of the bent section is K1, and the width of the main body section is K2, where K1:K2 = 1:

3. Preferably, the reaction element has an inclined draft angle θ2 in both the width and length directions, [0.5°≦θ2≦0.8°].

4. The reaction mixture according to any one of claims 1-3, characterized in that, The main body segment and the bending segment are integrally formed; or... The main body section and the bending section are separate components, and the bending section is detachably fixed to the end of the main body section.

5. The reaction mixture according to any one of claims 1-4, characterized in that, The main body segment is configured as a straight line; The bending segment is configured as a straight line, or, The bending segment is configured as an arc shape, and the concave direction of the arc-shaped bending segment is concave in the direction of its bending.

6. A reaction vessel, comprising a vessel body, characterized in that, The container body has an inner cavity for adaptably installing the reaction element according to any one of claims 1-5, and the inner cavity forms multiple partially sealed partitioned areas after the reaction element is installed.

7. The reaction vessel according to claim 6, characterized in that, The compatibility installation between the reaction vessel and the reaction components shall be adapted using one or more of the following installation ratios: (1) The length of the reaction element is L1, the height of the reaction vessel is L2, and L1:L2<90%; (2) The total straight width of the reaction vessel from the main body section to the bending section is L3, and the diameter of the reaction vessel is L4, where L3:L4<45%.

8. A cell-free reaction system, characterized in that, The reaction vessel includes the reaction element as described in any one of claims 1-5 and the reaction container as described in any one of claims 6-7, wherein the reaction element is installed perpendicular to the inner wall of the container body in the length direction and perpendicular to the bottom surface of the inner wall of the container body in the width direction.

9. The cell-free reaction system according to claim 8, characterized in that, The reaction element is fixed in the reaction vessel using any of the following methods: (1) The reaction element is vertically fixed to the inner wall of the container body in the length direction; (2) The reaction element is vertically fixed to the bottom surface of the inner cavity of the container body in the width direction; (3) The reaction element is vertically fixed to the inner wall of the container body in the length direction and vertically fixed to the bottom surface of the container body in the width direction. Preferably, the reaction element is provided with rounded corners between the inner wall of the container body and / or the top wall of the container base; Preferably, the number of the reactants is one or more; More preferably, the number of the reaction elements is set to 1 to 8.

10. A cell-free reaction method, characterized in that, Cellless reaction is performed using the cellless reaction system as described in claim 8 or 9; Preferably, the reaction method includes the following steps: Add reaction materials into the reaction vessel; The reaction vessel is placed at a preset tilt angle θ3; The driving reaction vessel rotates at a preset speed r, and the rotation process lasts for a preset time T; Preferably, the length of the reaction vessel is L1, and the height of the reaction material in the reaction vessel is L5, where L1:L5>200%.

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