Continuous culture device for phage induced protein directed evolution

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By designing a continuous culture device including a culture bottle group, a waste liquid bottle, a feed bottle, a peristaltic pump group and a hose compression switch assembly, the problems of cumbersome operation, high energy consumption and inhibition of bacterial growth in the prior art are solved, and the directional evolution of proteins is achieved quickly, automatic and continuous, and research efficiency and product synthesis efficiency are improved.

CN222556933UActive Publication Date: 2025-03-04潘艺 +2

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Patent Information

Application Number: CN202420642616.8
Authority / Receiving Office: CN · China
Patent Type: Utility models(China)
Current Assignee / Owner
Filing Date: 2024-03-29
Publication Date: 2025-03-04
Estimated Expiration: 2034-03-29

AI Technical Summary

Technical Problem

The existing phage-induced protein-oriented evolution devices have problems such as cumbersome operation, high energy consumption, large area, easy to contaminate and inhibition of bacterial growth.

Method used

A continuous culture device is designed, including a culture bottle group, waste liquid bottle, feed bottle, peristaltic pump group and hose compression switch assembly, which is connected through a silicone hose and replaced by a butterfly air filter. The hose compression switch is automatically controlled to open and break, realizing an automated and continuous protein-oriented evolution process.

Benefits of technology

It realizes the rapid, automatic and continuous progress of protein-oriented evolution, reduces manual intervention and repetitive work, shortens the time for mutation iteration accumulation, and improves bacterial growth and product synthesis efficiency.

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Abstract

The utility model belongs to the technical field of bioengineering, and discloses a continuous culture device for phage induced protein directed evolution, which comprises a first culture bottle and pipeline, a second culture bottle and pipeline, a third culture bottle and pipeline and a fourth culture bottle and pipeline, the peristaltic pump is respectively connected with the material supplementing bottle and the waste liquid bottle through the silica gel hose, and the butterfly-shaped air filter is respectively connected with the air compression pump and the emptying pump through the silica gel hose; a silica gel hose penetrates through a second hose pressing switch to be connected with a first three-way connector, the silica gel hose, a peristaltic pump, the second three-way connector and a third hose pressing switch to be connected with a second Luer female connector, and the second three-way connector penetrates through a fourth hose pressing switch to be connected with a waste liquid bottle through a silica gel hose; and the butterfly-shaped air filter is respectively connected with the air compression pump and the waste liquid bottle through the silica gel hose. The device is compact in structure, low in energy consumption, simple and convenient to operate and beneficial to thallus growth and product synthesis.

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Description

Technical Field

[0001] The utility model belongs to the technical field of bioengineering, and in particular relates to a continuous culture device for phage-induced protein directed evolution. Background Art

[0002] The natural evolution and natural selection of proteins will gradually move in a direction that is beneficial to the body. However, natural selection in the body is a very slow process. The in vitro directed evolution of proteins simulates the natural evolution of proteins in the laboratory. For the target protein, from mutation and recombination to final screening, the slow process of protein evolution is shortened by iterative accumulation. Through large-scale and efficient artificial screening, mutants that achieve the target performance are obtained. Its purpose is to design and apply genetic systems to selectively evolve target genes efficiently in cells. Whether it is selection or selection in the directed evolution of proteins, it often takes weeks or even months to complete from mutant construction, iterative accumulation to multiple rounds of screening. The intermediate process is interspersed with a lot of manual intervention, and a lot of work is used in repetitive work.

[0003] The existing phage-induced protein directed evolution device is composed of several shaking bottles and reagent bottles, as well as a constant temperature box and a magnetic stirrer. During the culture process, manual sampling, sampling and addition of inducers are performed.

[0004] The existing laboratory method for phage-induced directed evolution of proteins is: sterilize the shake flask and reagent bottle separately, then inoculate E. coli into the shake flask containing the culture medium in a biosafety cabinet, and then place it on a shaker for culture; after a certain period of culture, take a sample to determine the OD600 value of E. coli to determine the growth density of the engineered bacteria, then add phage, and then add the inducer arabinose after culturing the shake flask for a certain period of time, and continue to culture. At regular intervals, take out the shake flask to take out the phage sample for inspection until the culture is stopped after one week.

[0005] During the long process of the above-mentioned phage continuous induced protein directed evolution, it is necessary to take out the shake flask from the shaker and put it in the biosafety cabinet for sampling many times. The repetitive operation is very cumbersome and the shake flask is easily contaminated during the operation. At the same time, the shaker is used to culture the engineered bacteria and phage. The shaker is bulky and occupies a large space in the limited laboratory space. The shaker consumes a lot of electricity and is not energy-saving and environmentally friendly. In addition, the oxygen supply in the shake flask is very limited, and carbon dioxide, a byproduct of bacterial growth metabolism, is easily accumulated in the shake flask, inhibiting bacterial growth and product synthesis, and reducing the efficiency of phage continuous induced protein directed evolution. Utility Model Content

[0006] The utility model aims to solve at least one technical problem existing in the background technology, and provides a continuous culture device for phage-induced directed evolution of proteins, which has the characteristics of compact overall structure, low energy consumption and easy operation, so that the directed evolution of proteins can be carried out quickly, automatically and continuously, reducing human intervention, shortening the time for mutation iteration accumulation, reducing the repetitive workload of researchers, and being able to continuously supply oxygen to the bacteria in the shaking flask, reducing the accumulation of carbon dioxide, a metabolic product of bacterial growth, which is beneficial to bacterial growth and product synthesis.

[0007] In order to achieve the above technical objectives, the utility model adopts the following technical solutions:

[0008] A continuous culture device for phage-induced directed evolution of proteins, comprising a culture bottle group, a waste liquid bottle, a feed bottle, a peristaltic pump group and a hose compression switch assembly; the culture bottle group comprises a first culture bottle and a second culture bottle, the peristaltic pump group comprises four peristaltic pumps, and the hose compression switch assembly comprises four hose compression switches;

[0009] The connecting pipeline of the first culture bottle includes: connecting the first three-way joint through the silicone hose and the second peristaltic pump, the first three-way joint passes through the silicone hose through the first hose to press the switch to connect the first Luer female joint; connecting the feeding bottle through the silicone hose and the first peristaltic pump; connecting the waste liquid bottle through the silicone hose and the third peristaltic pump; connecting the air compression pump through the silicone hose and the second butterfly air filter; connecting the first butterfly air filter to exhaust waste gas through the silicone hose;

[0010] The connection pipeline of the second culture bottle includes: connecting the first three-way joint through the silicone hose passing through the second hose and pressing the switch; connecting the second three-way joint through the silicone hose and the fourth peristaltic pump, the second three-way joint through the silicone hose passing through the third hose and pressing the switch to connect the second Luer female joint, and at the same time, the second three-way joint through the silicone hose passing through the fourth hose and pressing the switch to connect the waste liquid bottle; connecting the air compression pump through the silicone hose and the third butterfly air filter; connecting the waste liquid bottle through the silicone hose and the fourth butterfly air filter;

[0011] The waste liquid bottle is connected to the fifth butterfly air filter through a silicone hose to exhaust the waste gas; the feed replenishment bottle is connected to the sixth butterfly air filter through a silicone hose to exhaust the waste gas.

[0012] Furthermore, the hose clamping switch assembly includes a bearing base, on which are arranged four hose clamping switches of the same structure, each of which includes a stepping motor, a slide rail support frame, a lead screw and a nut slide;

[0013] The slide rail support frame is fixedly connected to the upper end of the bearing base through fixing bolts and a vertical connecting plate. The slide rail support frame is a side U-shaped opening bracket, including an upper top plate and a lower bottom plate. A threaded hole is penetrated through the lower bottom plate. The top end of the screw rod passes through the threaded hole to fix the nut slide. The space between the nut slide and the upper top plate of the slide rail support frame is used to place a silicone hose.

[0014] The stepper motor is fixedly arranged at the bottom end of the bearing base, and the stepper motor is movably plug-connected to the bottom end of the lead screw; the stepper motor drives the lead screw to rotate, and the lead screw drives the nut slide to rise and fall in the side U-shaped opening groove of the slide rail support frame, thereby realizing the nut slide to squeeze and close or release and open the silicone hose.

[0015] Furthermore, an internal threaded hole is provided at the upper end of the screw rod, a mounting hole is provided on the nut slide, and the upper end of the screw rod and the nut slide are fixedly connected by limiting bolts through the internal threaded hole and the mounting hole;

[0016] An inner square hole is provided at the bottom end of the screw rod, and the driving end of the stepping motor is a square driving rod, which is movably inserted into the inner square hole at the bottom end of the screw rod.

[0017] Furthermore, the mounting hole formed on the nut slide is an inner stepped hole, and during installation, the upper end surface of the limit bolt is not higher than the upper surface of the nut slide.

[0018] Furthermore, the waste liquid bottle contains sodium hydroxide solution;

[0019] The first Luer female connector is used to connect to a first inoculation / sampling syringe; the second Luer female connector is used to connect to a second inoculation / sampling syringe.

[0020] Furthermore, the first peristaltic pump, the second peristaltic pump, the third peristaltic pump and the fourth peristaltic pump are all connected to the liquid inlet silicone hose and the liquid outlet silicone hose through a U-shaped silicone hose and a double-port silicone hose connector.

[0021] Furthermore, the feeding bottle comprises a feeding bottle body and a feeding bottle cover;

[0022] The feeding bottle cap is threadedly connected to the bottle mouth of the feeding bottle body through a first sealing silicone gasket, and two stainless steel hole covers are arranged in the feeding bottle cap, and two interfaces are arranged on the two stainless steel hole covers.

[0023] Furthermore, the first culture bottle comprises a first culture bottle body and a first culture bottle cap, the first culture bottle cap is screwed to the bottle mouth of the first culture bottle body through a second sealing silicone gasket; a five-way stainless steel hole cover is arranged in the first culture bottle cap, and five interfaces are arranged on the five-way stainless steel hole cover;

[0024] The upper port of one of the interfaces on the five-way stainless steel hole cover is connected to the air compression pump through a silicone hose and a second butterfly air filter, and the bottom short port is connected to a first silicone soft short tube, and the bottom end of the first silicone soft short tube is located at 1 / 2-1 / 3 of the height of the bottle body of the first culture bottle.

[0025] Furthermore, the second culture bottle comprises a second culture bottle body and a second culture bottle cover, the second culture bottle cover is screwed to the bottle mouth of the second culture bottle body through a third sealing silicone gasket; a first four-way stainless steel hole cover is arranged in the second culture bottle cover, and four interfaces are arranged on the first four-way stainless steel hole cover;

[0026] The upper port of one of the interfaces on the first four-way stainless steel hole cover is connected to the air compression pump through a silicone hose and a third butterfly air filter, and the bottom short port is connected to a second silicone soft short tube. The bottom end of the second silicone soft short tube is located at 1 / 2-1 / 3 of the height of the bottle body of the second culture bottle.

[0027] Furthermore, the waste liquid bottle includes a waste liquid bottle body and a waste liquid bottle cap, and the waste liquid bottle cap is threadedly connected to the bottle mouth of the waste liquid bottle body through a fourth sealing silicone gasket; a second four-way stainless steel hole cover is arranged in the waste liquid bottle cap, and 4 interfaces are arranged on the second four-way stainless steel hole cover.

[0028] Compared with the prior art, the beneficial effects produced by the utility model are:

[0029] (1) The utility model provides a continuous culture device for phage-induced protein directed evolution, comprising a culture bottle group, a waste liquid bottle, a feed bottle, a peristaltic pump group and a hose clamping switch assembly, wherein the above components are connected by a silicone hose, and the waste gas of the culture bottle is replaced by a butterfly air filter during the culture process, the waste gas of the waste liquid bottle is filtered and discharged after fire extinguishing and sterilization, and the waste gas of the feed bottle is filtered and discharged. The on-off of each silicone hose is automatically controlled by the hose clamping switch assembly, and the inoculation syringe or the sampling syringe is connected by the Luer female connector to achieve inoculation or regular sampling, eliminating the traditional bulky and energy-consuming shaking table. The whole device has the characteristics of compact structure, low energy consumption and easy operation, so that the directed evolution of protein can be carried out quickly, automatically and continuously, reducing manual intervention, shortening the time for mutation iteration accumulation, and reducing the repetitive workload of researchers. In addition, it can continuously supply oxygen to the bacteria in the culture bottle, reduce the accumulation of carbon dioxide, a metabolic product of bacterial growth, and is beneficial to bacterial growth and product synthesis;

[0030] (2) The hose compression switch assembly of the utility model is integrated with 4 hose compression switches. The hose compression switch gradually realizes the action of squeezing and closing or releasing and opening the silicone hose through the screw thread transmission. Since the opening and closing process is gradual for the liquid in the pipeline, the water hammer effect caused by the instantaneous opening and stopping of the traditional solenoid valve is avoided, which is beneficial to reduce the probability of damage to the pipeline. It can also avoid the valve body of the traditional switch valve directly contacting the liquid in the pipeline, causing contamination of the liquid and corrosion of the valve body structure, thereby extending the service life of the pipeline equipment. In addition, the hose compression switch is easy to process, simple and compact in size, and suitable for installation in a compact equipment space. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the three-dimensional structure of the continuous culture device of an embodiment of the utility model;

[0032] Figure 2 is a top view of the continuous culture device of an embodiment of the utility model;

[0033] Figure 3 This is a rear view of the continuous culture device of the embodiment of the utility model;

[0034] Figure 4 This is a schematic diagram of the structure of the first culture bottle and the feeding bottle connected in the embodiment of the utility model;

[0035] Figure 5 This is a schematic diagram of the structure of the first culture bottle and the second culture bottle connected to each other in an embodiment of the utility model;

[0036] Figure 6 This is a schematic diagram of the structure of the first culture bottle connected to the waste liquid bottle in an embodiment of the utility model;

[0037] Figure 7 This is a schematic diagram of the structure in which the second culture bottle is connected to the waste liquid bottle in an embodiment of the utility model;

[0038] Figure 8 It is a schematic diagram of the connection structure of the exhaust pipeline of the feeding bottle, the air intake pipeline and the exhaust pipeline of the first culture bottle, the air intake pipeline and the exhaust pipeline of the second culture bottle, and the exhaust pipeline of the waste liquid bottle in the embodiment of the utility model;

[0039] Fig. 9 This is a schematic structural diagram of a feeding bottle according to an embodiment of the utility model;

[0040] Fig.10 It is a schematic diagram of the explosion structure of the feeding bottle of the embodiment of the utility model;

[0041] Fig.11 This is a schematic structural diagram of a first culture bottle according to an embodiment of the utility model;

[0042] Fig.12 It is a schematic diagram of the explosion structure of the first culture bottle of the embodiment of the utility model;

[0043] Fig.13 This is a schematic diagram of the structure of the second culture bottle of the embodiment of the utility model;

[0044] Fig.14 It is a schematic diagram of the explosion structure of the second culture bottle in the embodiment of the utility model;

[0045] Fig.15 It is a structural schematic diagram of a waste liquid bottle according to an embodiment of the utility model;

[0046] Fig.16 It is a schematic diagram of the explosion structure of the waste liquid bottle of the embodiment of the utility model;

[0047] Fig.17 It is a structural schematic diagram of a hose compression switch assembly according to an embodiment of the utility model;

[0048] Fig.18 It is a schematic diagram of the exploded structure of the hose compression switch assembly of the embodiment of the utility model;

[0049] Fig.19 This is a schematic diagram of the structure of the first hose clamping switch of the embodiment of the utility model when it is in an open state;

[0050] Fig. 20 This is a schematic diagram of the structure of the first hose clamping switch of the embodiment of the utility model when it is in a closed state;

[0051] Fig.21 It is a schematic diagram of the explosion structure of the first hose compression switch of the embodiment of the utility model;

[0052] Explanation of the marks in the attached drawings: 1-chassis; 2-LCD touch screen of the programmable logic controller integrated machine; 3-power switch; 4-emergency stop switch; 5-first inoculation / sampling syringe; 6-second inoculation / sampling syringe; 7-air compression pump; 31-first peristaltic pump; 32-second peristaltic pump; 33-third peristaltic pump; 34-fourth peristaltic pump; 51-foot pad; 61-first butterfly air filter; 62-second butterfly air filter; 63-third butterfly air filter; 64-fourth butterfly air filter; 65-fifth butterfly air filter; 66-sixth butterfly air filter; 71-double-port silicone hose connection; 110-feeding bottle; 111-first silicone hose; 120-first culture bottle; 121-second Silicone hose; 122-first Luer female connector; 123-third silicone hose; 124-first three-way connector; 125-fourth silicone hose; 126-fifth silicone hose; 127-sixth silicone hose; 128-seventh silicone hose; 130-second culture bottle; 131-eighth silicone hose; 132-ninth silicone hose; 133-second three-way connector; 134-tenth silicone hose; 135 second Luer female connector; 136-eleventh silicone hose; 137-twelfth silicone hose; 138-thirteenth silicone hose; 139-fourteenth silicone hose; 140-waste bottle; 141-fifteenth silicone hose; 142-sixteenth silicone hose; 143-seventeenth silicone hose; 200-hose compression Switch assembly; 210-first hose clamping switch; 220 second hose clamping switch; 230-third hose clamping switch; 240-fourth hose clamping switch; 211-screw; 212-slide rail support frame; 213-limiting bolt; 214-nut slide; 215-fixing bolt; 251-stepping motor; 216-vertical connecting plate; 260-bearing base; 311-eighteenth silicone hose; 321-nineteenth silicone hose; 331-twentieth silicone hose; 311-U-shaped silicone hose; 411-feeding bottle body; 412-feeding bottle cover; 413-two-way stainless steel hole cover; 414-first sealing silicone gasket; 421-first culture bottle body; 422-first culture bottle cover; 423-five-way Stainless steel hole cover; 424-first silicone soft short tube; 425-second sealing silicone gasket; 426-first magnetic rotor; 431-second culture bottle body; 432-second culture bottle cap; 433-first four-way stainless steel hole cover; 434-second silicone soft short tube; 435-third sealing silicone gasket; 436-second magnetic rotor; 441-waste liquid bottle body; 442-waste liquid bottle cap; 443-second four-way stainless steel hole cover; 444-fourth sealing silicone gasket; 611-twenty-second silicone hose; 651-twenty-third silicone hose; 661-twenty-fourth silicone hose; 4131-two-way stainless steel hole cover first interface; 4132-two-way stainless steel hole cover second interface; 4231-five-way stainless steel hole cover first interface;4232-the second interface of the five-way stainless steel hole cover; 4233-the third interface of the five-way stainless steel hole cover; 4234-the fourth interface of the five-way stainless steel hole cover; 4235-the fifth interface of the five-way stainless steel hole cover; 4331-the first interface of the first four-way stainless steel hole cover; 4332-the second interface of the first four-way stainless steel hole cover; 4333-the third interface of the first four-way stainless steel hole cover; 4334-the fourth interface of the first four-way stainless steel hole cover; 4431-the first interface of the second four-way stainless steel hole cover; 4432-the second interface of the second four-way stainless steel hole cover; 4433-the third interface of the second four-way stainless steel hole cover; 4434-the fourth interface of the second four-way stainless steel hole cover. ; DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the utility model to clearly and completely describe the technical solutions in the embodiments of the utility model. Obviously, the described embodiments are only part of the embodiments of the utility model, not all of the embodiments. Based on the embodiments in the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0054] In the description of the present invention, it should be understood that the terms "coaxial", "bottom", "one end", "top", "middle", "the other end", "upper", "one side", "top", "inside", "front", "center", "both ends" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0055] In the present utility model, unless otherwise clearly stipulated and limited, the terms such as "installation", "setting", "connection", "fixation" and "screw-on" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integrated connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. Unless otherwise clearly defined, ordinary technicians in this field can understand the specific meanings of the above terms in the utility model according to the specific circumstances.

[0056] Combination Figure 1-Figure 21 As shown, an embodiment of the utility model provides a continuous culture device for phage-induced protein directed evolution, including a culture bottle group, a waste liquid bottle 140, a feed bottle 110, a peristaltic pump group and a hose compression switch assembly.

[0057] The culture bottle group includes a first culture bottle 120 and a second culture bottle 130 ( Figure 4 ); The peristaltic pump group includes four peristaltic pumps, namely a first peristaltic pump 31, a second peristaltic pump 32, a third peristaltic pump 33 and a fourth peristaltic pump 34 ( Figure 5 ); the hose clamping switch assembly 200 includes four hose clamping switches, namely: a first hose clamping switch 210, a second hose clamping switch 220, a third hose clamping switch 230 and a fourth hose clamping switch 240 ( Figure 5 and Figure 7 ).

[0058] like Figure 5 As shown, the first culture bottle 120 is connected to the first three-way connector 124 through the sixth silicone hose 127, the second peristaltic pump 32, and the fifth silicone hose 126 in sequence, the first three-way connector 124 is connected to the third silicone hose 123, the third silicone hose 123 passes through the first hose compression switch 210 to connect to the first Luer female connector 122, the first Luer female connector 122 is used to connect the first inoculation / sampling syringe 5, and is used to inoculate the first culture bottle 120 with Escherichia coli and multiple sampling during the culture process through the above-mentioned pipeline, wherein the volume of the first inoculation / sampling syringe 5 is selected to be 100 mL; at the same time, as Figure 4 As shown, the first culture bottle 120 is connected to the feeding bottle 110 through the second silicone hose 121, the first peristaltic pump 31, and the first silicone hose 111 in sequence. The feeding bottle 110 is used to hold the culture medium and transport the culture medium to the first culture bottle 120 through the above pipelines; at the same time, Figure 6 As shown, the first culture bottle 120 is connected to the waste liquid bottle 140 through the seventh silicone hose 128, the third peristaltic pump 33, and the fifteenth silicone hose 141 in sequence; at the same time, Figure 8 As shown, the first culture bottle 120 is connected to the air compression pump 7 through the fourteenth silicone hose 139, the second butterfly air filter 62, and the thirteenth silicone hose 138 in sequence, so as to introduce fresh air; at the same time, Figure 8 As shown, the first culture bottle 120 is connected to the first butterfly air filter 61 through the twenty-second silicone hose 611, which is used to discharge the carbon dioxide generated during the culture process to the outside to achieve gas renewal in the bottle.

[0059] Since the first culture bottle 120 is mainly used for culturing Escherichia coli, the growth of Escherichia coli requires culture medium and oxygen, and the byproducts of bacterial metabolism include carbon dioxide, etc. The accumulation of carbon dioxide will aggravate the acidification of the culture medium, thereby inhibiting the growth of Escherichia coli. Fresh air is compressed by the air compressor pump 7, introduced into the first culture bottle 120 through the thirteenth silicone hose 138 and the second butterfly air filter 62, and discharged at a position half the height of the bottle body to avoid a large number of bubbles in the culture medium. At the same time, the carbon dioxide accumulated in the first culture bottle 120 is discharged to the environment outside the bottle through the twenty-second silicone hose 611 and the first butterfly air filter 61, thereby realizing the gas renewal in the bottle, reducing the carbon dioxide content, which is beneficial to the growth of Escherichia coli.

[0060] Among them, all butterfly-shaped air filters used in the pipeline of the utility model are equipped with a filter membrane with a filter aperture of 0.22 μm, which can effectively intercept bacteria in the pipeline and prevent bacteria from contaminating the laboratory environment.

[0061] The second culture bottle 130 is mainly used to culture Escherichia coli and modified engineered phages, and the modified engineered phages are lysogenic phages; see Figure 5 As shown, the second culture bottle 130 is connected to the fourth silicone hose 125, and the fourth silicone hose 125 passes through the second hose compression switch 220 to connect to the first three-way connector 124, which is used to sequentially pass the Escherichia coli cultured in the first culture bottle 120 through the Figure 5 The sixth silicone hose 127, the nineteenth silicone hose 321 and the second peristaltic pump 32, the fifth silicone hose 126, the first three-way connector 124 and the fourth silicone hose 125 reach the second culture bottle 130 to perform the E. coli rehydration operation; at the same time, see Figure 7 As shown, the second culture bottle 130 is connected to the second three-way connector 133 through the eighth silicone hose 131, the fourth peristaltic pump 34, and the ninth silicone hose 132 in sequence, the second three-way connector 133 is connected to the tenth silicone hose 134, the tenth silicone hose 134 passes through the third hose compression switch 230 and is connected to the second Luer female connector 135, the second Luer female connector 135 is used to connect the second inoculation / sampling syringe 6, which is used to inject the modified engineered phage into the second culture bottle through the above-mentioned pipeline and multiple sampling during the culture process, wherein the volume of the second inoculation / sampling syringe 6 is selected to be 20mL, and at the same time, the second three-way connector 133 is also connected to the seventeenth silicone hose 143, the seventeenth silicone hose 143 passes through the fourth hose compression switch 240 and is connected to the waste liquid bottle 140, which is used to transfer the phage-containing bacterial liquid in the second culture bottle to the waste liquid bottle; at the same time, see Figure 8As shown, the second culture bottle 130 is connected to the air compression pump 7 through the twelfth silicone hose 137, the third butterfly air filter 63, and the eleventh silicone hose 136 in sequence for introducing fresh air. At the same time, the second culture bottle 130 is connected to the waste liquid bottle 140 through the sixteenth silicone hose 142, the fourth butterfly air filter 64, and the twenty-fifth silicone hose 144 in sequence for discharging carbon dioxide generated during the culture process into the waste liquid bottle, thereby realizing gas renewal in the second culture bottle 130.

[0062] The waste liquid bottle 140 contains 0.5-1 mol·L -1 The sodium hydroxide solution with a concentration of 400mL-900mL is used to inactivate the Escherichia coli or bacteriophage suspended in the waste gas discharged during the culture process before discharging it, so as to prevent the Escherichia coli or bacteriophage from being directly discharged with the waste gas and polluting the environment; the waste liquid bottle 140 is connected to the fifth butterfly air filter 65 through the twenty-third silicone hose 651, which is used to discharge the waste gas generated during the experimental culture process after inactivation and sterilization.

[0063] like Figure 6 As shown, the feeding bottle 110 is connected to the sixth butterfly air filter 66 via the twenty-fourth silicone hose 661, which is used to add air to the feeding bottle 110 to maintain the air pressure balance inside and outside the bottle, and filter the air through the sixth butterfly air filter 66 to prevent bacteria in the air from entering the feeding bottle 110.

[0064] See also Figure 4 As shown, the first peristaltic pump 31 is connected to the liquid inlet silicone hose and the liquid outlet silicone hose through the U-shaped silicone hose 311 and the double-port silicone hose connector 71, and the remaining peristaltic pumps are also connected to the liquid inlet silicone hose and the liquid outlet silicone hose through the corresponding U-shaped silicone hose and its double-port silicone hose connector 71.

[0065] See also Figure 17-Figure 18 As shown, the hose compression switch assembly 200 includes a supporting base 260 , on which four hose compression switches are arranged, namely: a first hose compression switch 210 , a second hose compression switch 220 , a third hose compression switch 230 and a fourth hose compression switch 240 .

[0066] The four hose compression switches have the same structure and are combined with Figure 17-Figure 21As shown, each hose clamping switch includes a stepper motor 251, a slide rail support frame 212, a screw rod 211 and a nut slide 214; the slide rail support frame 212 is fixedly connected to the upper end of the bearing base 260 by a fixing bolt 215 and a vertical connecting plate 216, the slide rail support frame 212 is a side U-shaped opening bracket, including an upper top plate and a lower bottom plate, a threaded hole 2121 is penetrated on the lower bottom plate of the slide rail support frame 212, the top end of the screw rod 211 passes through the threaded hole 2121 and is fixedly connected to the nut slide 214, and the space between the nut slide 214 and the upper top plate of the slide rail support frame 212 is used to place the silicone hose;

[0067] The stepper motor 251 is fixedly arranged at the bottom end of the supporting base 260, and the stepper motor 251 is movably plug-connected to the bottom end of the screw rod 211; when the stepper motor 251 drives the screw rod 211 to rotate, the screw rod 211 and the threaded hole 2121 form a screw thread transmission pair, so that the screw rod 211 can drive the nut slide 214 to rise and fall in the side U-shaped opening groove of the slide rail support frame 212, thereby realizing the nut slide 214 squeezing and closing or releasing and opening the silicone hose.

[0068] The upper end of the screw rod 211 is provided with an internal threaded hole, and the nut slide 214 is provided with a mounting hole. The upper end of the screw rod 211 and the nut slide 214 are fixedly connected by a limiting bolt 213 through the internal threaded hole and the mounting hole.

[0069] Furthermore, the mounting hole opened on the nut slide 214 is an inner stepped hole. During installation, the upper end surface of the limit bolt 213 is not higher than the upper surface of the nut slide 214, ensuring that the extruded end surface of the silicone hose extruded at the upper end of the nut slide 214 is always flat, so that the silicone hose is subjected to balanced force.

[0070] An inner square hole is provided at the bottom end of the screw rod 211, and the driving end of the stepper motor 251 is a square driving rod, which is movably inserted into the inner square hole at the bottom end of the screw rod 211; the screw rod 211 and the driving rod of the stepper motor 251 are connected through the square hole, and at the same time, the screw rod 211 and the driving rod of the stepper motor 251 are movably inserted, so that the driving rod of the stepper motor 251 can drive the screw rod 211 to rotate synchronously, and does not affect the screw rod 211 driving the nut slide 214 to move up and down.

[0071] The hose compression switch assembly 200 is integrated with four hose compression switches on the supporting base 260, which are respectively used to control the on and off of four silicone hoses. The overall size is small and is particularly suitable for installation in a compact equipment space.

[0072] See also Figure 9-10As shown, the feeding bottle 110 includes a feeding bottle body 411 and a feeding bottle cover 412, and the feeding bottle cover 412 is threadedly screwed to the bottle mouth of the feeding bottle body 411 through a first sealing silicone gasket 414, and a two-way stainless steel hole cover 413 is arranged in the feeding bottle cover 412, and the two-way stainless steel hole cover 413 is provided with a two-way stainless steel hole cover first interface 4131 and a two-way stainless steel hole cover second interface 4132.

[0073] The bottom long port of the first interface 4131 of the two-way stainless steel hole cover and the bottom short port of the second interface 4132 of the two-way stainless steel hole cover are inserted into the feeding bottle body 411; the upper port of the first interface 4131 of the two-way stainless steel hole cover is connected to the first silicone hose 111, and the upper port of the second interface 4132 of the two-way stainless steel hole cover is connected to the sixth butterfly air filter 66 through the twenty-fourth silicone hose 661.

[0074] See also Figure 11-Figure 12 As shown, the first culture bottle 120 includes a first culture bottle body 421 and a first culture bottle cover 422, the first culture bottle cover 422 is threadedly screwed to the bottle mouth of the first culture bottle body 421 through a second sealing silicone gasket 425, and a five-way stainless steel hole cover 423 is arranged in the first culture bottle cover 422, and the five-way stainless steel hole cover 423 is provided with a five-way stainless steel hole cover first interface 4231, a five-way stainless steel hole cover second interface 4232, a five-way stainless steel hole cover third interface 4233, a five-way stainless steel hole cover fourth interface 4234, and a five-way stainless steel hole cover fifth interface 4235.

[0075] The bottom long port of the first interface 4231 of the five-way stainless steel hole cover, the bottom short port of the second interface 4232 of the five-way stainless steel hole cover, the bottom short port of the third interface 4233 of the five-way stainless steel hole cover, the bottom short port of the fourth interface 4234 of the five-way stainless steel hole cover, and the bottom long port of the fifth interface 4235 of the five-way stainless steel hole cover are all inserted into the first culture bottle body 421.

[0076] Among them, the bottom short port of the fourth interface 4234 of the five-way stainless steel hole cover is connected to the first silicone soft short tube 424, and the bottom end of the first silicone soft short tube 424 is located at the half of the height of the first culture bottle body 421; the upper port of the first interface 4231 of the five-way stainless steel hole cover is connected to the sixth silicone hose 127; the upper port of the second interface 4232 of the five-way stainless steel hole cover is connected to the second butterfly air filter 62 through the fourteenth silicone hose 139; the upper port of the third interface 4233 of the five-way stainless steel hole cover is connected to the first butterfly air filter 61 through the twenty-second silicone hose 611; the upper port of the fifth interface 4235 of the five-way stainless steel hole cover is connected to the seventh silicone hose 128; the fourth interface 4231 of the five-way stainless steel hole cover is connected to the sixth silicone hose 127; the upper port of the second interface 4232 of the five-way stainless steel hole cover is connected to the second butterfly air filter 62 through the fourteenth silicone hose 139; the upper port of the third interface 4233 of the five-way stainless steel hole cover is connected to the first butterfly air filter 61 through the twenty-second silicone hose 611; the upper port of the fifth interface 4235 of the five-way stainless steel hole cover is connected to the seventh silicone hose 128; The upper port of the first culture bottle 34 is connected to the fourteenth silicone hose 139, and the fourteenth silicone hose 139 is connected to the second butterfly air filter 62, the thirteenth silicone hose 138 and the air compression pump 7 in sequence, so as to introduce the fresh air compressed by the air compression pump 7 into the first culture bottle body 421 at a position half of the height of the bottle body and discharge it, so as to avoid a large number of bubbles generated when the culture medium is inserted; a first magnetic rotor 426 is placed in the first culture bottle body 421, and a controllable and constantly changing external magnetic field (the external magnetic field can be provided by an electromagnetic wire or a magnetic part driven to rotate by a motor) is arranged outside the first culture bottle body 421, so as to control the rotation of the first magnetic rotor 426 placed in the first culture bottle body 421, stir the solution in the culture bottle, mix it and increase the dissolved oxygen content.

[0077] See also Figure 13-14 As shown, the second culture bottle 130 includes a second culture bottle body 431 and a second culture bottle cover 432, the second culture bottle cover 432 is threadedly screwed to the bottle mouth of the second culture bottle body 431 through a third sealing silicone gasket 435, and a first four-way stainless steel hole cover 433 is arranged in the second culture bottle cover 432, and the first four-way stainless steel hole cover 433 is provided with a first four-way stainless steel hole cover first interface 4331, a first four-way stainless steel hole cover third interface 4333, a first four-way stainless steel hole cover second interface 4332 and a first four-way stainless steel hole cover fourth interface 4334.

[0078] Among them, the bottom long port of the first interface 4331 of the first four-way stainless steel hole cover, the bottom short port of the second interface 4332 of the first four-way stainless steel hole cover, the bottom short port of the third interface 4333 of the first four-way stainless steel hole cover and the bottom short port of the fourth interface 4334 of the first four-way stainless steel hole cover are all inserted into the second culture bottle body 431, the bottom short port of the third interface 4333 of the first four-way stainless steel hole cover is connected to the second silicone soft short tube 434, and the bottom end of the second silicone soft short tube 434 is located at half the height of the bottle body of the second culture bottle body 431.

[0079] The upper port of the first interface 4331 of the first four-way stainless steel hole cover is connected to the eighth silicone hose 131; the upper port of the second interface 4332 of the first four-way stainless steel hole cover is connected to the fourth silicone hose 125; the upper port of the third interface 4333 of the first four-way stainless steel hole cover is connected to the twelfth silicone hose 137, and the twelfth silicone hose 137 is connected to the third butterfly air filter 63, the eleventh silicone hose 136 and the air compression pump 7 in sequence, so as to introduce the fresh air compressed by the air compression pump 7 into the position half of the height of the second culture bottle body 431 The fourth interface 4334 of the first four-way stainless steel hole cover is connected to the sixteenth silicone hose 142; a second magnetic rotor 436 is placed in the second culture bottle body 431, and a controllable and constantly changing external magnetic field (the external magnetic field can be provided by an electromagnetic wire or a magnetic part driven by a motor to rotate) is arranged outside the second culture bottle body 431 to control the rotation of the second magnetic rotor 436 placed in the second culture bottle body 431, so as to stir the solution in the culture bottle to mix it and increase the dissolved oxygen content.

[0080] See also Figure 15-16 As shown, the waste liquid bottle 140 includes a waste liquid bottle body 441 and a waste liquid bottle cap 442, and the waste liquid bottle cap 442 is threadedly screwed to the bottle mouth of the waste liquid bottle body 441 through a fourth sealing silicone gasket 444, and a second four-way stainless steel hole cover 443 is arranged in the waste liquid bottle cap 442, and a second four-way stainless steel hole cover first interface 4431, a second four-way stainless steel hole cover second interface 4432, a second four-way stainless steel hole cover third interface 4433 and a second four-way stainless steel hole cover fourth interface 4434 are arranged on the second four-way stainless steel hole cover 443.

[0081] Among them, the bottom long port of the first interface 4431 of the second four-way stainless steel hole cover, the bottom long port of the second interface 4432 of the second four-way stainless steel hole cover, the bottom short port of the third interface 4433 of the second four-way stainless steel hole cover and the bottom long port of the fourth interface 4434 of the second four-way stainless steel hole cover are all inserted into the glass waste liquid bottle body 441.

[0082] The upper port of the first interface 4431 of the second four-way stainless steel hole cover is connected to the fifteenth silicone hose 141; the upper port of the second interface 4432 of the second four-way stainless steel hole cover is connected to the twenty-fifth silicone hose 144; the upper port of the third interface 4433 of the second four-way stainless steel hole cover is connected to the twenty-third silicone hose 651; the upper port of the fourth interface 4434 of the second four-way stainless steel hole cover is connected to the seventeenth silicone hose 143.

[0083] In the embodiment of the present utility model, Figure 1As shown, it also includes a chassis 1, and the culture bottle group, waste liquid bottle 140, feeding bottle 110, peristaltic pump group, hose compression switch assembly, and various silicone hoses and other components are all integrated on the chassis 1.

[0084] In addition, the chassis 1 is also provided with a liquid crystal touch screen 2, a power switch 3 and an emergency stop switch 4 of a programmable logic controller all-in-one, and foot pads 51 are provided at the four corners of the bottom of the chassis 1; the power switch 3 is used to control the on and off of the power supply required by the device; the emergency stop switch 4 can quickly cut off the power supply when the device operates abnormally, thereby playing a power-off protection role.

[0085] The above description is only an embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the scope of application of the present invention shall be included in the protection scope of the present invention.

Claims

1. A continuous culture device for phage-induced directed evolution of proteins, characterized in that: It includes a culture bottle group, a waste liquid bottle, a feed bottle, a peristaltic pump group and a hose compression switch assembly; the culture bottle group includes a first culture bottle and a second culture bottle, the peristaltic pump group includes 4 peristaltic pumps, and the hose compression switch assembly includes 4 hose compression switches; The connecting pipeline of the first culture bottle includes: connecting the first three-way joint through the silicone hose and the second peristaltic pump, the first three-way joint passes through the silicone hose through the first hose to press the switch to connect the first Luer female joint; connecting the feeding bottle through the silicone hose and the first peristaltic pump; connecting the waste liquid bottle through the silicone hose and the third peristaltic pump; connecting the air compression pump through the silicone hose and the second butterfly air filter; connecting the first butterfly air filter to exhaust waste gas through the silicone hose; The connection pipeline of the second culture bottle includes: connecting the first three-way joint through the silicone hose passing through the second hose and pressing the switch; connecting the second three-way joint through the silicone hose and the fourth peristaltic pump, the second three-way joint through the silicone hose passing through the third hose and pressing the switch to connect the second Luer female joint, and at the same time, the second three-way joint through the silicone hose passing through the fourth hose and pressing the switch to connect the waste liquid bottle; connecting the air compression pump through the silicone hose and the third butterfly air filter; connecting the waste liquid bottle through the silicone hose and the fourth butterfly air filter; The waste liquid bottle is connected to the fifth butterfly air filter through a silicone hose to exhaust the waste gas; the feed replenishment bottle is connected to the sixth butterfly air filter through a silicone hose to exhaust the waste gas.

2. The continuous culture device according to claim 1, characterized in that: The hose clamping switch assembly includes a bearing base, on which are arranged four hose clamping switches of the same structure, each of which includes a stepping motor, a slide rail support frame, a lead screw and a nut slide; The slide rail support frame is fixedly connected to the upper end of the bearing base through fixing bolts and a vertical connecting plate. The slide rail support frame is a side U-shaped opening bracket, including an upper top plate and a lower bottom plate. A threaded hole is penetrated through the lower bottom plate. The top end of the screw rod passes through the threaded hole to fix the nut slide. The space between the nut slide and the upper top plate of the slide rail support frame is used to place a silicone hose. The stepper motor is fixedly arranged at the bottom end of the bearing base, and the stepper motor is movably plug-connected to the bottom end of the lead screw; the stepper motor drives the lead screw to rotate, and the lead screw drives the nut slide to rise and fall in the side U-shaped opening groove of the slide rail support frame, thereby realizing the nut slide to squeeze and close or release and open the silicone hose.

3. The continuous culture device according to claim 2, characterized in that: The upper end of the screw rod is provided with an internal threaded hole, and the nut slide is provided with a mounting hole. The upper end of the screw rod and the nut slide are fixedly connected by limiting bolts through the internal threaded hole and the mounting hole. An inner square hole is provided at the bottom end of the screw rod, and the driving end of the stepping motor is a square driving rod, which is movably inserted into the inner square hole at the bottom end of the screw rod.

4. The continuous culture device according to claim 3, characterized in that: The mounting hole formed on the nut slide is an inner stepped hole. During installation, the upper end surface of the limit bolt is not higher than the upper surface of the nut slide.

5. The continuous culture device according to any one of claims 1 to 4, characterized in that: The waste liquid bottle contains sodium hydroxide solution; The first Luer female connector is used to connect to a first inoculation / sampling syringe; the second Luer female connector is used to connect to a second inoculation / sampling syringe.

6. The continuous culture device according to any one of claims 1 to 4, characterized in that: The first peristaltic pump, the second peristaltic pump, the third peristaltic pump and the fourth peristaltic pump are all connected to the liquid inlet silicone hose and the liquid outlet silicone hose through the U-shaped silicone hose and the double-port silicone hose connector.

7. The continuous culture device according to any one of claims 1 to 4, characterized in that: The feeding bottle comprises a feeding bottle body and a feeding bottle cover; The feeding bottle cap is threadedly connected to the bottle mouth of the feeding bottle body through a first sealing silicone gasket, and two stainless steel hole covers are arranged in the feeding bottle cap, and two interfaces are arranged on the two stainless steel hole covers.

8. The continuous culture device according to any one of claims 1 to 4, characterized in that: The first culture bottle comprises a first culture bottle body and a first culture bottle cap, wherein the first culture bottle cap is threadedly connected to the bottle mouth of the first culture bottle body through a second sealing silicone gasket; a five-way stainless steel hole cover is arranged in the first culture bottle cap, and five interfaces are arranged on the five-way stainless steel hole cover; The upper port of one of the interfaces on the five-way stainless steel hole cover is connected to the air compression pump through a silicone hose and a second butterfly air filter, and the bottom short port is connected to a first silicone soft short tube, and the bottom end of the first silicone soft short tube is located at 1 / 2-1 / 3 of the height of the bottle body of the first culture bottle.

9. The continuous culture device according to any one of claims 1 to 4, characterized in that: The second culture bottle comprises a second culture bottle body and a second culture bottle cap, the second culture bottle cap is screwed to the bottle mouth of the second culture bottle body through a third sealing silicone gasket; a first four-way stainless steel hole cover is arranged in the second culture bottle cap, and four interfaces are arranged on the first four-way stainless steel hole cover; The upper port of one of the interfaces on the first four-way stainless steel hole cover is connected to the air compression pump through a silicone hose and a third butterfly air filter, and the bottom short port is connected to a second silicone soft short tube. The bottom end of the second silicone soft short tube is located at 1 / 2-1 / 3 of the height of the bottle body of the second culture bottle.

10. The continuous culture device according to any one of claims 1 to 4, characterized in that: The waste liquid bottle comprises a waste liquid bottle body and a waste liquid bottle cap, wherein the waste liquid bottle cap is screwed to the bottle mouth of the waste liquid bottle body through a fourth sealing silicone gasket; a second four-way stainless steel hole cover is arranged in the waste liquid bottle cap, and four interfaces are arranged on the second four-way stainless steel hole cover.