High-throughput automatic monoclonal screening and picking workstation

Abstract

The invention belongs to the technical field of bacterial culture, and discloses a high-throughput automatic monoclonal screening and picking workstation, which comprises a protective box for protection and isolation, and a workbench is arranged in the protective box; the plate stack module comprises a rotating plate stack arranged at the bottom end of the workbench, and a plurality of columns of culture dishes are arranged on the rotating plate stack in a lifting manner; the transfer module is arranged on the workbench and used for transferring culture dishes; the pipetting module comprises a pipetting assembly and a material storage assembly, and the pipetting assembly is used for transferring strains in the culture dish into the material storage assembly; the identification module is used for identifying and positioning the strains in the culture dish and then controlling the pipetting assembly to accurately transfer the strains. The device is simple in structure and flexible and convenient to use, high-throughput screening and picking of strains can be fully automatically carried out in a clean closed environment, manual intervention is not needed, the screening efficiency is high, strain pollution caused by manual intervention is avoided, the screening precision is high, the screening cost is reduced, and experiments are greatly facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial culture, and in particular relates to a high-throughput automated monoclonal screening and picking workstation. Background Art

[0002] In experimental bacteriology, bacteria are usually manipulated, stored and transported in the form of cultures. However, these cultures may contain genetic variation or simply contaminants, so the isolation, cultivation and picking of monoclonal colonies are key operations in microbiological research.

[0003] The scalability and fast turnaround time of high-throughput gene synthesis have promoted the progress of genetics, medicine and various biotechnology fields. A key step of high-throughput gene synthesis is to connect the gene fragments to the cloning vector or expression vector and transform them into competent cells, then spread the cells on the culture container for culture, and finally pick several single clones for sequencing to obtain a 100% correct gene clone. Conventional solid plate culture based on manual coating and picking of colonies usually consumes a lot of time, labor and material resources. Although the existing high-throughput cloning colony picking system has multiple cloning picking needles with rotating functions, which can be used to continuously transfer colonies to the target plate, the system can only load one colony source at a time, does not support the fully automatic picking of colonies from multiple culture equipment, and has a relatively low application scope. Therefore, although these two systems can perform high-throughput cloning picking, they cannot meet the needs of fully automated multiple clone picking for a large number of gene constructions.

[0004] Therefore, it is necessary to develop a fully automatic colony picking device with comprehensive functions that can be applied to more application scenarios. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a high-throughput automated monoclonal screening and picking workstation, which is mainly used for high-throughput screening of correct clones of a large number of samples downstream of high-throughput gene construction and colony rearrangement to meet the needs of different sequencing schemes.

[0006] To achieve the above object, the present invention provides a high-throughput automated monoclonal screening and picking workstation, comprising:

[0007] A protective box, used for protection and isolation, wherein a workbench is provided in the protective box;

[0008] The plate stack module comprises a rotating plate stack rotatably arranged at the bottom of the workbench, and a plurality of culture dishes arranged in a vertical row are arranged on the rotating plate stack; a lifting assembly corresponding to the culture dishes is arranged at the bottom of the workbench, and is used to drive the culture dishes to be lifted and lowered on both sides of the workbench;

[0009] A transfer module is arranged on the workbench and corresponds to the culture dish on the top of the workbench;

[0010] A pipetting module, the pipetting module comprising a correspondingly arranged pipetting component and a storage component, the pipetting component transfers the bacterial strains in the culture dish into the storage component;

[0011] The identification module is arranged on the pipetting component and is used to identify and locate the bacterial species in the culture dish and then control the precise transfer of the pipetting component.

[0012] Preferably, the rotating plate stack is provided with a plurality of storage positions spaced axially apart, the stacked culture dishes are placed on the storage positions, and the lifting assembly is arranged corresponding to the storage positions to drive the culture dishes placed on the storage positions to be lifted and lowered.

[0013] Preferably, any of the storage positions is vacant without the culture dish, and the remaining storage positions are used to place the culture dishes, and the lifting assembly is arranged corresponding to the vacant storage position and an adjacent storage position.

[0014] Preferably, the lifting assembly includes two lifting frames, one of which is arranged corresponding to the vacant storage stack, and the other of which is arranged corresponding to the storage stack adjacent to the vacant storage stack; a lifting plate for longitudinal lifting is arranged on the lifting frame, and the lifting plate supports the culture dish to be lifted and lowered.

[0015] Preferably, the transfer module includes a first manipulator and a second manipulator disposed on the workbench, the first manipulator being used to transfer the culture dish between adjacent storage stacks, and the second manipulator being used to remove the cover of the culture dish after inoculation and to recover the glass beads in the cover.

[0016] Preferably, the pipetting assembly includes a movable track arranged in the protective box, a pipetting block is slidably arranged on the movable track, a plurality of pipette guns are movably arranged at the bottom end of the pipetting block, and after the culture dish is transferred to the selection position on the workbench, the bacterial strain in the culture dish is transferred to the storage assembly.

[0017] Preferably, the material storage assembly includes a plurality of deep-well plates arranged on the workbench as culture vessels, and the bacterial species in the culture vessels are identified and located by the identification module and then transferred to the deep-well plates through the pipette.

[0018] Preferably, the material storage assembly includes a recovery box and a plurality of consumable boxes arranged on the workbench, wherein the consumable box stores the pipette tips for replacement, and the pipette tips of the pipette are recovered into the recovery box after being used once.

[0019] Preferably, the recognition module includes a recognition frame arranged on the movable track, and a distance sensor for measuring and a shooting camera for image recognition are movably arranged on the recognition frame.

[0020] Preferably, a calibration position is provided on the workbench for performing calibration and positioning after the pipette tip is replaced with a new one.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects: the present invention discloses a high-throughput automated monoclonal screening and picking workstation, the equipment operates in a fully enclosed operating environment protection box, the process is fully automated, the risk of sample contamination caused by the environment or human errors is reduced, and the colonies can be rearranged according to actual needs, which reduces manual intervention, significantly improves experimental efficiency, and greatly reduces manpower and time costs; when working, the plate stack module is used to store culture dishes filled with bacterial strains, the culture dishes are stacked and placed on the rotating plate stack, and the fully loaded culture dishes are driven to rise to the workbench through the lifting component, and the selected culture dishes are recovered again, and the transfer module realizes the transfer of the culture dishes, and then fully automated screening and picking can be performed; the pipetting module can operate automatically, and the identification module thereon screens and locates the bacterial strains in the culture dish, and then controls the pipetting component of the pipetting module to pick the corresponding bacterial strains and transfer them to the discharging component, completing the automated high-throughput screening and picking of bacterial strains.

[0022] The invention has a simple structure and is flexible and convenient to use. It can automatically perform high-throughput screening and picking of bacterial strains in a clean closed environment without manual intervention. The screening efficiency is high, bacterial strain contamination caused by manual intervention is avoided, the screening accuracy is high, the screening cost is reduced, and the experiment is greatly facilitated. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0024] Figure 1 This is an axial view of the high-throughput automated monoclonal screening and picking workstation of the present invention;

[0025] Figure 2 A top view of the working plate of the present invention;

[0026] Figure 3 It is a side view of the plate stack module of the present invention;

[0027] Figure 4 A top view of the plate stack module of the present invention;

[0028] Figure 5This is a view of the first manipulator axis of the present invention;

[0029] Figure 6 This is an axial view of the liquid transfer module of the present invention;

[0030] Figure 7 A photograph of a culture dish taken by the camera of the present invention;

[0031] Figure 8 This is a photo of the conventional monoclonal selection culture dish before spot picking in the present invention;

[0032] Fig. 9 Automatically identify the colonies to be selected in the conventional monoclonal selection culture dish of the present invention and circle them in red;

[0033] Fig.10 This is a photo of the conventional monoclonal selection culture dish after spot picking in the present invention;

[0034] Fig.11 This is a photo of the culture dish before spot selection in the blue-white colony selection of the present invention;

[0035] Fig.12 In the blue-white colony selection of the present invention, the colonies to be selected are automatically identified in the culture dish and circled in red;

[0036] Fig.13 This is a photo of the blue-white colony in the culture dish after spot selection in the present invention;

[0037] In the figure: 1. Protection box; 2. Workbench; 3. Rotating plate stack; 4. Culture dish; 5. Storage stack; 6. Lifting rack; 7. Lifting plate; 8. Loading hole; 9. Return hole; 10. First manipulator; 11. Second manipulator; 12. Capping; 13. Moving track; 14. Pipette block; 15. Pipette gun; 16. Picking position; 17. Deep well plate; 18. Recovery box; 19. Consumables box; 20. Pipette tip; 21. Identification rack; 22. Distance sensor; 23. Shooting camera; 24. Manual channel; 25. Bead dumping box; 26. Calibration position. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] Reference Figure 1-Figure 7 As shown, this embodiment provides a high-throughput automated monoclonal screening and picking workstation, comprising:

[0041] A protection box 1 is used for protection and isolation, and a workbench 2 is provided inside the protection box 1;

[0042] The plate stack module comprises a rotating plate stack 3 rotatably arranged at the bottom of the workbench 2, and a plurality of culture dishes 4 arranged in a vertical row are arranged on the rotating plate stack 3; a lifting assembly corresponding to the culture dishes 4 is arranged at the bottom of the workbench 2, and is used to drive the culture dishes 4 to rise and fall on the upper and lower sides of the workbench 2;

[0043] A transfer module is arranged on the workbench 2 and is arranged corresponding to the culture dish 4 on the top of the workbench 2;

[0044] A pipetting module, the pipetting module includes a correspondingly arranged pipetting component and a storage component, and the pipetting component transfers the bacterial species in the culture dish 4 to the storage component;

[0045] The identification module is set on the pipetting component and is used to identify and locate the bacterial species in the culture dish, and then control the precise transfer of the pipetting component.

[0046] The present invention discloses a high-throughput automated monoclonal screening and picking workstation. The equipment runs in a fully enclosed operating environment protection box 1, and the process is fully automated, which reduces the risk of sample contamination caused by the environment or human errors. The colonies can be rearranged according to actual needs, which reduces human intervention, significantly improves experimental efficiency, and greatly reduces manpower and time costs. When working, the plate stack module is used to store culture dishes 4 filled with bacterial strains, and the culture dishes 4 are stacked and placed on a rotating plate stack 3. The fully loaded culture dishes 4 are driven to rise to a workbench 2 through a lifting component, and the selected culture dishes 4 are recovered again, and the transfer module realizes the transfer of the culture dishes 4, and then fully automated screening and picking can be performed. The pipetting module can run automatically, and the identification module thereon screens and locates the bacterial strains in the culture dishes 4, and then controls the pipetting component of the pipetting module to pick the corresponding bacterial strains and transfer them to the discharging component, thereby completing automated high-throughput screening and picking of bacterial strains. The invention has a simple structure and is flexible and convenient to use. It can automatically perform high-throughput screening and picking of bacterial strains in a clean closed environment without manual intervention. The screening efficiency is high, bacterial strain contamination caused by manual intervention is avoided, the screening accuracy is high, the screening cost is reduced, and the experiment is greatly facilitated.

[0047] Furthermore, the protective box 1 of this embodiment is made of transparent material, which can facilitate observation of the screening and picking process.

[0048] Furthermore, the outer wall of the protective box 1 is provided with a shading structure that can be opened and closed to facilitate adjusting the light intensity when screening and picking the strains that need shading, thereby creating a proper lighting environment.

[0049] Furthermore, the protective box 1 of the present embodiment is provided with an openable and closable box door, which can form a sealed isolation environment when the box door is closed.

[0050] Furthermore, the protective box 1 of this embodiment is provided with a mechanism for disinfecting the protective box 1, so as to facilitate sterilization of the protective box 1 before and after the experiment, and prevent miscellaneous bacteria in the protective box 1 from affecting the progress of the experiment.

[0051] Furthermore, the sterilization mechanism of this embodiment can select ultraviolet lamp or ozone generating device, and the disinfection method and principle are conventional rotation direction, which will not be repeated here.

[0052] Furthermore, the workbench 2 of the present embodiment can be pushed out from the door opening of the protective box 1 under the drive of internal power, which is convenient for cleaning and also convenient for inspection and maintenance of the structure thereon.

[0053] Furthermore, a manual passage 24 is provided on the side of the workbench 2 facing the door of the protection box 1 for manually placing the culture dish 4.

[0054] Further optimization scheme, a plurality of storage stacks 5 arranged at axial intervals are provided on the rotating plate stack 3, the stacked culture dishes 4 are placed on the storage stacks 5, and the lifting assembly is arranged corresponding to the storage stacks 5, driving the culture dishes 4 placed on the storage stacks 5 to be lifted and lowered. The rotating plate stack 3 rotates under the drive of the driving element, and the storage stacks 5 are arranged in a ring shape on the rotating plate stack 3. A plurality of culture dishes 4 containing bacterial strains are inverted and stacked and stuck on the storage stacks 5; when in use, the lifting assembly drives the culture dishes 4 containing bacterial strains to move to the workbench 2 through the loading hole 8 on the workbench 2, and then the culture dishes 4 are transferred to the designated position for screening and picking through the transfer module, and the processed culture dishes 4 fall back from the return hole 9 to the storage stack 5, realizing automatic processing.

[0055] In a further optimized solution, any storage position 5 is vacant without a culture dish 4, and the remaining storage positions 5 are used to place culture dishes 4, and the lifting assembly is set corresponding to the vacant storage position 5 and an adjacent storage position 5. In this embodiment, 9 storage positions 5 are set on the rotating stack, and each storage position 5 can hold 12 standard culture dishes 4. However, in actual use, one storage position 5 is vacant without a culture dish 4, and the remaining 8 storage positions 5 are filled with culture dishes 4 containing bacterial strains. After the culture dishes 4 containing bacterial strains are screened and picked, the used culture dishes 4 are returned to the vacant storage positions 5, so that the bacterial strains in the culture dishes 4 can be fully automatically screened and picked without manual intervention.

[0056] Furthermore, the storage stack 5 of the present embodiment is surrounded by a plurality of vertical rods with adjustable spacing, which can be conveniently adapted to different culture dishes 4, thereby improving applicability.

[0057] Further optimization scheme, the lifting assembly includes two lifting racks 6, one lifting rack 6 is set corresponding to the vacant storage stack 5, and the other lifting rack 6 is set corresponding to the storage stack 5 adjacent to the vacant storage stack 5; the lifting rack 6 is provided with a longitudinal lifting plate 7, and the lifting plate 7 lifts the culture dish 4. The lifting assembly includes two lifting racks 6, and the two lifting racks 6 are respectively set corresponding to the feeding hole 8 and the return hole 9. One lifts the culture dish 4 containing the bacterial strain to gradually rise to the workbench 2 through the lifting plate 7, which is convenient for the transfer module to transfer, and the other lift rack 6 supports the used culture dish 4 through the lifting plate 7, and then gradually descends, which is convenient for the recovery of the used culture dish 4.

[0058] Further optimization scheme, the transfer module includes a first manipulator 10 and a second manipulator 11 arranged on the workbench 2, the first manipulator 10 is used to transfer the culture dish 4 between adjacent storage stacks 5, and the second manipulator 11 is used to remove the cover 12 for the inoculated culture dish 4. The first manipulator 10 and the second manipulator 11 adopt the electric gripper method, which can identify culture dishes 4 of different sizes and grasp them stably; when working, the first manipulator 10 grasps the culture dish 4 entering from the feed port, then flips the culture dish 4 and transfers it to the selection position 16 for screening and picking, and then transfers the picked culture dish 4 to the position of the return port; the second manipulator 11 removes the cover 12 of the culture dish 4, then stably grasps the cover 12 left at the feed port, and transfers it to the top of the bead dumping box 25 to flip it to recover the coated glass beads that may remain on the cover 12, and then store it, and finally cover it back to the culture dish 4 that has been inoculated for recycling, so as to realize automatic operation.

[0059] Further optimization scheme, the pipetting assembly includes a moving track 13 arranged in the protective box 1, a pipetting block 14 is slidably arranged on the moving track 13, and a plurality of pipetting guns 15 are movably arranged at the bottom of the pipetting block 14. After the culture dish 4 is transferred to the selection position 16 on the workbench 2, the strains in the culture dish 4 are transferred to the storage assembly. The moving track 13 is arranged along the X-axis of the work box, and the pipetting block 14 slides on the moving track 13 to realize the movement of the X-axis. The pipetting gun 15 that can move along the Y-axis is arranged on the pipetting block 14. At the same time, the pipetting gun 15 can be lifted and lowered longitudinally, so that the suction port on the pipetting gun 15 can be moved in three-dimensional space. The recognition module identifies and locates the strains in the culture dish 4 on the selection position 16, and then controls the pipetting gun 15 to move to the specified position, and transfers the selected strains to the discharge assembly to complete the inoculation of the strains.

[0060] Furthermore, the present embodiment is provided with 8 independently arranged pipette guns 15, which automatically adapt to the selection height, have a variable pitch direction speed of 300 mm / s, and a z-axis speed of 150 mm / s, and are used for colony selection, including various microorganisms, bacteria, yeast, bacteriophages, molds, etc.

[0061] Furthermore, after the culture dish 4 is placed on the selection position 16 , it can be automatically rotated so that the position of the culture dish 4 can be adjusted.

[0062] A further optimized solution is that the material storage component includes a plurality of deep-well plates 17 as culture vessels arranged on the workbench 2. After the bacterial species in the culture dish 4 are identified and located by the identification module, they are transferred to the deep-well plates 17 through the pipette 15; the material storage component includes a recovery box 18 and a plurality of consumable boxes 19 arranged on the workbench 2. The consumable box 19 stores pipette tips 20 for replacement of the pipette 15. The pipette tips 20 of the pipette 15 are discarded in the recovery box 18 after being used once. The deep-well plate 17 is placed on the workbench 2, on which are provided a number of grooves for culturing bacterial strains. During operation, the bacterial strains selected from the culture dish 4 are inoculated into the deep-well plate 17 through the pipette 15, so as to facilitate subsequent culturing. At the same time, a number of consumable boxes 19 are also provided on the workbench 2, and the consumable boxes 19 are arranged side by side with the deep-well plate 17, and the consumable boxes 19 store consumables for picking bacterial strains therein, so as to facilitate the selection by the pipette 15. After one picking, the pipette 15 moves to the top of the recovery box 18, and the pipette tip 20 at the end of the pipette 15 automatically falls off and is discarded in the recovery box 18, and then returns to the top of the consumable box 19, and a new pipette tip 20 is installed in the consumable box 19, and the bacterial strains are picked again, so that a new pipette tip is used for each bacterial strain, and there is no interference.

[0063] Furthermore, the bottom end of the recycling box 18 of the present embodiment is connected to a trash can placed in the protective box for recycling discarded suction tips 20. The used suction tips 20 are placed in the recycling box 18 and fall into the trash can, which is convenient for recycling.

[0064] Furthermore, six 24 / 48 / 96 deep-well plates 17 can be placed on the workbench 2, and other plates (such as PCR 96-well plates or 384-well plates) can be compatible by replacing the base adapter.

[0065] Furthermore, 6 boxes of pipette tips 20 consumables can be placed on the workbench 2.

[0066] Furthermore, the deep well plate 17 and the consumable box 19 can be pulled out along the y-axis direction to facilitate the placement and removal of the deep well plate 17 and the consumables.

[0067] Further optimizing the scheme, the recognition module includes a recognition frame 21 arranged on the moving track 13, and the recognition frame 21 is movably provided with a distance sensor 22 for measurement and a shooting camera 23 for image recognition. The recognition frame 21 is slidably connected to the moving track 13, and can be easily moved. The distance sensor 22 is used to measure the distance to the culture dish 4, which is convenient for identification and positioning; the shooting camera 23 is used to shoot the bacterial plaque on the culture dish 4, which can distinguish different colors of bacterial plaques, and identify qualified single colonies in high definition, automatically image and select colonies, or manually select and cancel colonies.

[0068] Furthermore, the identification frame 21 and the moving block move separately, and the bacterial species in the culture dish 4 are photographed and positioned in advance, thereby improving work efficiency.

[0069] Furthermore, the moving speed of the moving block and the identification frame 21 is 600 mm / s, and the position accuracy is 0.02 mm.

[0070] Further optimizing the scheme, a calibration position 26 is provided on the workbench 2 for calibration and positioning after the pipette gun 15 is replaced with a new pipette tip 20. The calibration position 26 is used to calibrate the angle of the pipette tip 20, so as to facilitate accurate picking of the colonies on the culture dish 4 and avoid picking errors caused by uneven surface or different heights of the solid culture medium.

[0071] Furthermore, a bead dumping box 25 is provided on the workbench 2 to facilitate storage of the beads in the culture dish 4 .

[0072] Workflow:

[0073] a. Manually place the culture dishes 4 into the storage stacks 5 one by one, with the bottom of the culture dishes 4 facing upward and the lid facing downward, taking care to prevent the beads inside the bottom lid from contacting the bacterial colonies on the culture medium, until 12 culture dishes 4 are installed in 8 of the storage stacks 5.

[0074] b. After ensuring that each culture dish 4 is correctly placed, start the system, and the lifting component will lift the culture dish 4 in the storage stack 5 upward along the feed port. When it is lifted to a certain height and the sensor senses the culture dish 4, the lifting component stops working and waits for the next operation.

[0075] c. The first manipulator 10 rotates to the position of the culture dish 4 at the feed port, and accurately grips the culture dish 4, automatically flips it 180° to turn the surface of the solid culture medium from downward to upward, and then places the culture dish 4 at the selection position 16.

[0076] d. At the same time, the second manipulator 11 moves to the position of the feed inlet culture dish 4, and accurately clamps the cover 12, transfers it to the top of the bead dumping box 25, and automatically turns 180 degrees to dump the beads in the cover 12. After the beads are dumped, they are stored in the designated location.

[0077] e. The selection position 16 automatically rotates 180° horizontally to transfer the culture dish 4 on the left to the right position, and the camera 23 above focuses on the colonies on the surface of the solid culture medium to automatically image and select colonies, or manually select or deselect colonies.

[0078] The following are the colony automatic identification process and standards:

[0079] First, identify all the white cells in the image.

[0080] Next, determine the distance between cells.

[0081] If cells are too close to each other, the device may interfere with other cells when picking out the target cell. Therefore, the algorithm will first calculate the distance between all cells, and use the distance between the cell and the nearest cell as the distance attribute of the cell. If the distance attribute of a cell has an attribute value of 3000, it means that there are no other cells within 3000 of the cell. By setting the threshold of the distance attribute, all sparse cells greater than the threshold are screened out.

[0082] Next, set the selection boundaries.

[0083] If the cell is very close to the border of the culture dish 4, the device will not be able to pick it up, and the suction head 20 may hit the inner wall of the culture dish 4. And at the border of the culture dish 4, the cell imaging is easily disturbed by external light sources. Therefore, only cells within a certain border range are selected. Set a border attribute for the cell, which measures its distance to the center of the image. By setting a threshold for the border attribute, all center cells smaller than the threshold are screened out.

[0084] Second, screen the cells for roundness.

[0085] In culture dish 4, there will be many cells that have not differentiated, which does not meet the selection conditions. Multiple cells have not differentiated, and they will be identified as one cell, but the shape is similar to a gourd, etc. A roundness attribute is set for all cells that meet the spacing and boundary conditions, which measures the degree to which the cell outline tends to be round. The larger the roundness, the rounder the cell outline. Multiple cells that have not differentiated can be screened out by setting a high roundness threshold.

[0086] Secondly, determine the maximum and minimum cell areas of the selected clones

[0087] All cells that meet the spacing, boundary, and roundness are arranged in ascending order by area. If the minimum area is set to 0.5 and the maximum area is set to 0.9, all cells in the 50%-90% range of the cell queue are selected as qualified cells for selection. The reason for setting the minimum area and maximum area is that cells with too small or too large areas are not target cells for selection and cloning.

[0088] Set the number of selections and determine the exclusion of bubbles

[0089] If 8 cells are selected, all cells that meet the requirements of spacing, boundary, roundness, and area are sorted from large to small, and then judged and output in sequence. For the first cell in the queue, judge whether it is a bubble. If it is not a bubble, output it, otherwise skip it; for the second cell in the queue, judge whether it is a bubble. If it is not a bubble, output it, otherwise skip it. And so on, until the target number of cells is output, or there are no more cells in the cell queue.

[0090] f. The distance sensor 22 detects the height of the colony to be picked in the culture dish 4, and the moving block drives the pipette gun 15 to automatically load the tip 20 and then transfers it to the calibration position 26 of the tip 20 to calibrate the angle of the tip 20.

[0091] g. After the angle of the pipette tip 20 is calibrated, the moving block carries the pipette tip 20 to the top of the culture dish 4 at the selection position 16. By precisely controlling the descending height, the pipette tip 20 is placed on the colonies to be picked one by one, and then is simultaneously transferred to the top of the deep-well plate 17 containing the liquid culture medium, and the different colonies are simultaneously punched into their respective wells. Finally, the used pipette tip 20 is discarded into the recovery box 18, or the pipette tip 20 is directly punched into the respective wells of the deep-well plate 17.

[0092] h..The picking position 16 performs another 180° horizontal rotation to transfer the culture dish 4 on the right side that has completed colony picking to the left side, and the first manipulator 10 moves the culture dish 4 to the return port position, and then the second manipulator 11 transfers the cap 12 to the return port to cap the corresponding culture dish 4.

[0093] i. After the sensor senses the culture dish 4, the lifting plate 7 corresponding to the return hole 9 will drop to the depth of a culture dish 4 to achieve the recovery of the used culture dish 4.

[0094] j. After the system is started, the system will repeat the operation of step b1 until all culture dishes 4 are processed. Finally, the deep-well plate 17 containing the bacterial colonies is manually transferred to the shaking table for shaking and expansion.

[0095] Equipment biochemical application verification

[0096] The present invention uses plasmids constructed from different gene synthesis products to carry out competent cell transformation and culture, and uses a culture dish 4 containing colonies to carry out high-throughput clone identification and selection verification.

[0097] Specific experimental steps:

[0098] 1. Enzymatically ligate the synthesized gene fragment to the linearized vector, and then transform the ligated product into 100uLDH5a or Top10 competent E. coli, add 500uLSOC culture medium, shake at 37℃ 200rpm for 1 hour, and take different amounts (100uL-500uL) on solid culture dish 4 (solid culture medium thickness not exceeding 4mm), and use glass beads (glass beads diameter not exceeding 5mm) for coating.

[0099] 2. Turn the culture dish 4 together with the glass beads upside down and place it in an incubator at 37°C for overnight culture (14-16 hours).

[0100] 3. Place the culture dish 4 with bacterial plaque (different densities) into the material unit of the device of the present invention (the device has been sterilized by UV for at least 30 minutes in advance). The transfer process should be as gentle and careful as possible to prevent the glass beads at the bottom of the culture dish 4 from contacting the solid culture medium and bacterial colonies on the top.

[0101] 4. Place the deep well plate 17 containing 200uL culture medium (containing resistance) in each well and the pipette tip 20 at the designated position of the present invention.

[0102] 5. Start the equipment, and the equipment automatically completes the identification of the colonies in all culture dishes 4, and picks 4 or 6 colonies that meet the requirements in morphology from each culture dish 4 and puts them into the deep well plate 17.

[0103] 6. Manually take out the deep well plate 17 and seal it, then transfer it to a shaker at 220 rpm and 37°C for 2-3 hours.

[0104] 7. Take 5uL of bacterial solution for bacterial solution PCR verification.

[0105] 8. Add another 1 mL of culture medium to the 96-deep-well plate 17 bacterial solution, continue shaking at 220 rpm and 37°C for 12 hours, extract the plasmid, and perform Sanger sequencing verification.

[0106] In the description of the present invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention, 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.

[0107] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-throughput automated monoclonal screening and picking workstation, characterized in that: include: A protection box (1) for protection and isolation, wherein a workbench (2) is arranged inside the protection box (1); The plate stack module comprises a rotating plate stack (3) rotatably arranged at the bottom end of the workbench (2), and a plurality of culture dishes (4) arranged in a vertical row are arranged on the rotating plate stack (3); a lifting assembly corresponding to the culture dishes (4) is arranged at the bottom end of the workbench (2), and is used to drive the culture dishes (4) to be lifted and lowered on the upper and lower sides of the workbench (2); A transfer module is arranged on the workbench (2) and is arranged corresponding to the culture dish (4) on the top of the workbench (2); A liquid transfer module, the liquid transfer module comprising a corresponding liquid transfer component and a material storage component, the liquid transfer component transfers the bacterial strain in the culture dish (4) into the material storage component; The identification module is arranged on the pipetting component and is used to identify and locate the bacterial species in the culture dish and then control the precise transfer of the pipetting component.

2. The high-throughput automated monoclonal screening and picking workstation according to claim 1, characterized in that: The rotating plate stack (3) is provided with a plurality of storage positions (5) arranged at axial intervals, the culture dishes (4) arranged in a stacked manner are placed on the storage positions (5), and the lifting assembly is arranged corresponding to the storage positions (5) to drive the culture dishes (4) placed on the storage positions (5) to be lifted or lowered.

3. The high-throughput automated monoclonal screening and picking workstation according to claim 2, characterized in that: Any of the storage stacks (5) is vacant and does not contain the culture dish (4), while the remaining storage stacks (5) contain the culture dishes (4), and the lifting assembly is arranged corresponding to the vacant storage stack (5) and an adjacent storage stack (5).

4. The high-throughput automated monoclonal screening and picking workstation according to claim 2, characterized in that: The lifting assembly comprises two lifting racks (6), one of the lifting racks (6) being arranged corresponding to the vacant storage stack (5), and the other of the lifting racks (6) being arranged corresponding to the storage stack (5) adjacent to the vacant storage stack (5); a lifting plate (7) for longitudinal lifting is arranged on the lifting rack (6), and the lifting plate (7) lifts the culture dish (4) up and down.

5. The high-throughput automated monoclonal screening and picking workstation according to claim 2, characterized in that: The transfer module comprises a first manipulator (10) and a second manipulator (11) arranged on the workbench (2), wherein the first manipulator (10) is used to transfer the culture dish (4) between adjacent storage stacks (5), and the second manipulator (11) is used to remove the cover (12) of the culture dish (4) after inoculation and to recover the glass beads in the cover (12).

6. The high-throughput automated monoclonal screening and picking workstation according to claim 1, characterized in that: The pipetting assembly comprises a movable track (13) arranged in the protective box (1), a pipetting block (14) is slidably arranged on the movable track (13), and a plurality of pipetting guns (15) are movably arranged at the bottom end of the pipetting block (14). After the culture dish (4) is transferred to the selection position (16) on the workbench (2), the bacterial strain in the culture dish (4) is transferred to the storage assembly.

7. The high-throughput automated monoclonal screening and picking workstation according to claim 6, characterized in that: The material storage assembly comprises a plurality of deep-well plates (17) arranged on the workbench (2) as culture vessels. The bacterial species in the culture vessels (4) are identified and located by the identification module and then transferred to the deep-well plates (17) via the pipette (15).

8. The high-throughput automated monoclonal screening and picking workstation according to claim 6, characterized in that: The material storage component comprises a recovery box (18) and a plurality of consumable boxes (19) arranged on the workbench (2); the consumable box (19) stores the pipette tips (20) of the pipette gun (15) for replacement; the pipette tips (20) of the pipette gun (15) are recovered into the recovery box (18) after being used once.

9. The high-throughput automated monoclonal screening and picking workstation according to claim 6, characterized in that: The recognition module comprises a recognition frame (21) arranged on the movable track (13), and a distance sensor (22) for measuring and a shooting camera (23) for image recognition are movably arranged on the recognition frame (21).

10. The high-throughput automated monoclonal screening and picking workstation according to claim 8, characterized in that: The workbench (2) is provided with a calibration position (26) for performing calibration and positioning after the pipette gun (15) is replaced with a new pipette tip (20).

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