Robotic platform for the culturing and evolution of microbial organisms

Abstract

A robotic culturing system may include a plurality of culturing units, a robotic arm configured to transfer culture tubes between the plurality of culturing units, a culture tube storage rack, and a media dispensing station. The system may also include a processor and memory storing instructions that, when executed, cause the system to carry out a method that may include measuring parameters of a culture within a tube, analyzing the sensor data to determine a growth status, and determining, based on the analyzed growth status, whether propagation of the microbial population should occur, and in response to determining that propagation should occur, controlling the robotic arm to receive growth media into an empty new tube, controlling the robotic arm to transfer an aliquot of the microbial population from a current tube to the new tube, and initiating a new batch of microbial culture in the new tube.

Description

Docket No. 24636-778 WO 1ROBOTIC PLATFORM FOR THE CULTURING AND EVOLUTION OF MICROBIAL ORGANISMSRELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 713,497, filed October 29, 2024, which is incorporated, in its entirety, by this reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM057089 awarded by the National Institutes of Health. The government has certain rights in the invention.SUMMARY

[0003] In some example embodiments, there may be provided systems, methods, and articles of manufacture for a robotic platform including one or more culturing units.

[0004] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The accompanying drawings, which are incorporated in and constitute a part of this specification, show certain aspects of the subject matter disclosed herein and, together with the description, help explain some of the principles associated with the disclosed implementations.Docket No. 24636-778 WO 1

[0006] FIG. 1A depicts aspects of a batch culture laboratory evolution technique performed by the robotic platform, in accordance with embodiments disclosed herein;

[0007] FIG. IB depicts a plot of batches 1-10 of cultured microbe versus microbial fitness, in accordance with embodiments disclosed herein;

[0008] FIG. 1C depicts an example implementation of the robotic platform system, in accordance with embodiments disclosed herein;

[0009] FIG. ID depicts an example implementation of the culturing system, in accordance with embodiments disclosed herein;

[0010] FIG. IE depicts another example implementation of the robotic platform system, in accordance with embodiments disclosed herein;

[0011] FIG. IF depicts another example implementation of the robotic platform system, in accordance with embodiments disclosed herein;

[0012] FIG. 2A depicts an example perspective view of the culturing unit, in accordance with embodiments disclosed herein; FIG. 2B depicts a cutaway view of the culturing unit of FIG. 2A, in accordance with embodiments disclosed herein;

[0013] FIG. 2C depicts an exploded view of the culturing unit of FIG. 2A, in accordance with embodiments disclosed herein;

[0014] FIG. 2D depicts a system diagram of a culturing system, in accordance with embodiments disclosed herein;

[0015] FIG. 3A depicts an embodiment of a robotic platform system in accordance with embodiments disclosed herein;

[0016] FIG. 3B depicts a robotic arm with a gripper module and pipettor module of a robotic platform system, in accordance with embodiments disclosed herein;Docket No. 24636-778 WO 1

[0017] FIG. 3C depicts an example of a growth media dispensing station of a robotic platform system, in accordance with embodiments disclosed herein;

[0018] FIG. 3D depicts aspects of an example culture media flow used in a robotic platform system, in accordance with embodiments disclosed herein;

[0019] FIG. 4A depicts a block diagram of process control methods and data structures used by a robotic platform system to perform aspects of batch culture laboratory evolution techniques, in accordance with embodiments disclosed herein;

[0020] FIG. 4B depicts a block diagram of process control methods and data structures for machine control used by a robotic platform system to perform aspects of batch culture laboratory evolution techniques, in accordance with embodiments disclosed herein;

[0021] FIG. 4C depicts a block diagram of process control methods and data structures for experimental control used by a robotic platform system to perform aspects of batch culture laboratory evolution techniques, in accordance with embodiments disclosed herein;

[0022] FIGS 5A depicts a process for using a robotic platform for automatic culture laboratory evolution, in accordance with embodiments disclosed herein;

[0023] FIG. 5B depicts aspects of a measuring process, in accordance with embodiments disclosed herein;

[0024] FIGS. 5C-1 and 5C-2 depict aspects of a propagation process, in accordance with embodiments disclosed herein;

[0025] FIG. 6A depicts a graph of culture densities versus time for a batch culture evolution under constant culture media definitions, in accordance with embodiments disclosed herein;Docket No. 24636-778 WO 1

[0026] FIG. 6B depicts aspects of growth rates and stress levels versus the sequential batch # under a tolerization selection pressure package, in accordance with embodiments disclosed herein;

[0027] FIG. 6C depicts aspects of culture density versus time under a non-native growth condition selection pressure package, in accordance with embodiments disclosed here;

[0028] FIG. 7A depicts an example perspective view of the culturing unit,

[0029] FIG. 7B depicts the culturing unit of FIG. 7A with the outer housing removed,

[0030] FIG. 7C depicts an exploded view of the culturing unit of FIG. 7A,

[0031] FIG. 7D depicts a cutaway view of the culturing unit of FIG. 7A along with views of subassemblies and components,

[0032] FIG. 8 depicts a system diagram of a culturing system, in accordance with embodiments disclosed herein.DETAILED DESCRIPTION

[0033] Adaptive laboratory evolution (ALE) is a process that cultures organisms, such as microbes, under controlled conditions to evolve desired traits, such as improved growth rate, tolerance to growth environments, production of target compounds, and / or the like. This in a sense mimics natural evolution, but ALE accelerates the evolution in a laboratory setting by subjecting the organisms to conditions that impose selection pressures for the desired traits.

[0034] FIG. 1A depicts an overview of the batch culture laboratory evolution technique (ALE) 10 performed by the robotic platform disclosed herein. At FIG. 1A, periodically during batch culture ALE, an aliquot (e.g., sample) of a microbial population is taken from a first batch 11 and propagated to a second batch 12 that provides a fresh growth media 14 to continue growth. When this occurs, selection pressures at the second batch 12 may be adjusted by for exampleDocket No. 24636-778 WO 1 changing parameters, such as growth media composition, temperature, and / or the like. Routinely, an aliquot of the microbial population is taken from the second batch 12 and propagated to a third batch 13 that provides a fresh growth media 16 to continue growth. When this occurs, selection pressures at the third batch 13 may be adjusted by, for example, changing parameters, such as growth media composition, temperature, and / or the like. The propagation and growth may continue for many batches. For example, FIG. IB depicts a plot of batches 1-10 of a cultured microbe versus microbial fitness, which may be associated with various desired traits. As can be seen at FIG. IB, after a variable amount of time growing under a selection pressure, increases in fitness may be observed.

[0035] In some embodiments, there is provided a robotic platform configured to perform variable duration, moderate volume batch culture ALE of microbes. Adaptive evolution may be induced through high-frequency culture monitoring by the robotic platform, propagation of microbial populations to fresh growth media, and control and manipulation of selection pressures,.

[0036] FIG. 1C shows a picture of an example implementation of the robotic platform system 100. The robotic platform system includes one or more culturing systems 110, a robot arm 112, tube storage racks 114, a media dispenser 116, and a robot arm tool(s) 118 including a gripper and pipettor located on a surface 102, which may provide for repeatable and modular positioning for components, such as through the use of T-slots.

[0037] As described herein, the culturing systems 110 may include one or more modular culturing units configured to receive and maintain culture tubes containing microbial populations. Each culturing unit may provide controlled environmental conditions such as temperature, aeration, and mixing through integrated heaters, magnetic stirrers, and associated control electronics. The culturing systems 110 may further include sensors to measure aspects of theDocket No. 24636-778 WO 1 culture, such as culture density, which may be derived from light scattering, and absorbance measurements at one or more wavelengths and angles. In some embodiments, each culturing unit is removably mounted to a base plate that provides both electrical power and data communication.

[0038] The robotic arm 112 may include a multi-axis robotic manipulator configured for precise, repeatable movement within the workspace defined by the surface 102. The robot arm 112 may include encoders and joint sensors that provide positional feedback to a system controller. The arm’s motion may be guided by a kinematic model referenced to a calibrated coordinate system of the platform. In some embodiments, the robot arm 112 may also incorporate a vision system or proximity sensors. The robot ami may be programmed to perform automated workflows such as transferring culturing containers, such as tubes or plates, or other labware, pipetting, dispensing, or discarding materials in response to process control software instractions.

[0039] The tube storage racks 114 may be configured to securely hold a plurality of empty or filled culture tubes in known, indexed positions that are accessible to the robot arm 112. Each rack may include mechanical alignment features such as keyed recesses, slots, or tapered guides to ensure consistent placement of tubes. The racks may also include barcodes or fiducial markers that allow the system to identify individual tubes or rack locations during operation. In some embodiments, the racks may include temperature-controlled compartments or removable trays to preserve samples or reagents at a desired temperature while awaiting processing. The tube storage racks 114 may further include integrated sensors or switches that detect whether a tube position is occupied, allowing the system controller to track inventory and prevent collisions during robotic transfer.

[0040] The media dispenser 116 may include one or more dispensing stations configured to deliver precise volumes of liquid growth media or supplements into culture tubes. The dispenserDocket No. 24636-778 WO 1 may employ syringe pumps, peristaltic pumps, or other metering mechanisms connected to a network of tubing and valves for controlled delivery of media components. Each pump may be associated with a specific growth media component or supplement, allowing dynamic formulation of different media compositions during culturing or propagation operations. The media dispenser 116 may further include reservoirs or bottles that store bulk media solutions, and may be connected to the platform’s control system for automated activation. In some embodiments, the dispenser includes rinse or priming lines to prevent cross-contamination between media components. The dispenser head or outlet ports may be located at fixed coordinates accessible to the robot arm 112, enabling the arm to position culture tubes beneath the correct dispensing line during operation.

[0041] The robot arm tools 118 may include interchangeable end effectors attachable to the distal end of the robot arm 112. Example tools include a gripper configured to grasp and manipulate culture tubes and other laboratory objects, and a micropipettor configured to aspirate and dispense small liquid volumes. The micropipettor may be configured to interface with standard disposable pipette tips and may include electronic actuation for precise volume control. In some embodiments, an automated tool-changing mechanism may allow the robot arm 112 to switch between the gripper and micropipettor during an experimental sequence. Each tool may have associated calibration data stored in the system controller so that when the tool is mounted, the robot automatically adjusts its motion parameters to account for tool geometry and weight. In some embodiments, the distal end of the arm may include an effector system that includes both a micropipette and a gripper, such as depicted herein. For example, tool adapter 338 of FIG. 3B may simultaneously carry the gripper module 320 and a micropipettor.

[0042] In some embodiments, the robotic platform system 100 may be configured such that each of its major components, including the culturing systems 110, tube storage racks 114,Docket No. 24636-778 WO 1 media dispenser 116, and robot arm tool(s) 118, are located in fixed, known positions and orientations relative to one another on surface 102. The fixed positioning may allow for precise robotic movement and automated workflows. Each component may be mounted to the surface 102 using alignment features such as dowel pins, slots, or registration fixtures to ensure positional repeatability. The robot arm 112 may be calibrated to a known coordinate system defined with respect to itself, such as its base, or with respect to the surface 102, to move culture tubes, pipette tips, and other objects between components with sub-millimeter accuracy.

[0043] The robotic platform system 100 may further include one or more fiducial markers, optical reference targets, or other alignment features 108 located at predetermined positions on the surface 102 and / or on the individual components. These may be detected by a vision system 106 associated with the robot arm 112, such as a camera mounted on the end effector or the robot base, to verify system alignment and automatically recalibrate component positions if necessary. The vision system may also detect barcodes, QR codes, or other optical identifiers 104 on the culture tubes, racks, and consumables to ensure proper tracking of materials throughout the culturing and propagation processes.

[0044] In some embodiments, the surface 102 may include a grid-based reference system, such as an embedded coordinate track 107, to assist in the spatial organization of the platform. The grid may be encoded into the robot’s control software, such that each equipment element (culturing system 110, media dispenser 116, tube rack 114, etc) is assigned a unique coordinate or “slot” in the workspace. This structured arrangement simplifies both software mapping and physical setup of the platform, allowing for reproducible assembly and reconfiguration when scaling or modifying the system.Docket No. 24636-778 WO 1

[0045] The robotic arm 112 may be a collaborative-type arm with multiple degrees of freedom, capable of precise, repeatable motions across a large working envelope. The robot arm’s tool center point may be dynamically adjusted within the control software depending on which of the robot arm tool(s) 118 (e.g., gripper or micropipettor) is currently attached. The system may include an automated tool changer that allows the robot to interchange between end effectors during operation without manual intervention. This capability allows the platform to execute sequences of handling, pipetting, and dispensing actions without interruption, further reducing operator labor.

[0046] To facilitate safe and reliable operation, the robotic platform system 100 may include sensors for detecting the presence, position, and orientation of each culture tube and consumable. These may include capacitive proximity sensors, load cells, or vision-based presence detectors located in the culturing systems 110, storage racks 114, and media dispenser 116. In some implementations, the surface 102 may include embedded identification or contact sensors that confirm when a rack or component is correctly seated in its designated position, thereby preventing misalignment that could interfere with automated motion paths.

[0047] FIG. ID depicts an example implementation of the culturing system 110. The culturing system comprises a culturing unit 120 with a container into which a culture tube 122 is inserted. A base surface of the culturing unit 120 may be coupled (e.g., snap fit) into a culturing unit base plate 124. The culturing unit base plate 124 may be configured to accommodate a plurality of culturing units (which in this example is 8 culturing units although other quantities may be accommodated as well). The culturing unit 120 is configured to monitor, test, and / or perform other functions associated with culturing a microbe. The culturing unit is configured to enable batch culturing as the culture tubes can be rapidly, individually, and robotically loaded andDocket No. 24636-778 WO 1 unloaded with the robotic arm 112 and gripper (e.g., fingers) 118. The culturing unit 120 may include sensors to perform measurements and / or light dosing using for example four wavelengths at specific angles, such as 180 degrees (sensors and light emitters on opposite sides of the sample and directed at each other through the sample) and 90 degrees (sensors and light emitters on adjacent sides and / or the emitter configured to emit light through the sample at an angle of about 90 degrees with respect to the direction of the sensor). In some embodiments, other quantities or wavelengths and angles may be implemented within the culturing module in addition to or in place of the 180 degree and 90 degree arrangements. Moreover, the culturing unit may include a heater, magnetic stirrers, temperature sensor, pH sensor, and stirring sensor, etc., as discussed herein. There may also be included a source of power and a data interface 126 through which a culturing unit and / or a culturing system may be powered, and its functionality controlled. In some embodiments, power and control of each culturing unit 120 may be provided through the strip 124.

[0048] In some embodiments, the culturing unit 120 may be removably coupled to the culturing system 110 through one or more coupling features 199A and 199B located on the culturing system. The culturing unit 120 may include corresponding mating features that engage with the coupling features 199 A and 199B to secure the culturing unit in place. In one example, the coupling may be implemented using a snap-fit mechanism, in which resilient tabs or detents on the culturing unit 120 engage with corresponding recesses, ridges, or latches on the coupling features 199 A and 199B. This configuration allows the culturing unit 120 to be easily installed or removed without the need for tools while maintaining a stable and repeatable attachment during operation. In some embodiments, the couplings 199A and 199B may be electrical couplings through with data communication and electrical power may be passed from or through the base 124 to the culturing unit 120.Docket No. 24636-778 WO 1

[0049] In some embodiments, the coupling between the culturing unit 120 and the coupling features 199A and 199B may be achieved magnetically. For example, magnets may be embedded within or adjacent to the coupling features 199 A and 199B, and corresponding magnetic elements or ferromagnetic inserts may be located on the mating portions of the culturing unit 120 or vice versa. This configuration may allow the culturing units to be attached and detached, while magnetic attraction ensures consistent seating and retention during operation. In some examples, the magnetic coupling may be combined with mechanical locating pins or alignment keys to prevent rotational movement or lateral shifting once installed.

[0050] The coupling features 199 A and 199B may also serve as alignment structures that ensure each culturing unit 120 is positioned in a known and repeatable spatial relationship (position and orientation) with respect to the culturing system 110 and the platform coordinate system. For example, in some embodiments, the coupling features may include locating bosses, keyed channels, or alignment pins that interface with complementary recesses or slots on the culturing unit and / or vice versa. This mechanical registration allows the system controller and robot arm 112 to operate based on known, fixed spatial coordinates, enabling automated placement and retrieval of culture tubes with high positional accuracy.

[0051] In some embodiments, the culturing system 110 may have a linear configuration in which one or more, such as multiple culturing units 120 are coupled along a single axis, forming a line of individual units. This arrangement simplifies robotic access and cable routing, allowing the robot arm 112 to service each unit sequentially along a defined linear path.

[0052] In some embodiments, multiple culturing units 120 may be coupled together in two orthogonal directions to form a two-dimensional (2D) array of culturing units. In such a configuration, each unit is coupled to the culturing system 110 and to adjacent units through theDocket No. 24636-778 WO 1 coupling features 199 A and 199B or similar connection points, allowing for a modular and scalable arrangement. The resulting 2D array may form a grid or matrix layout that increases throughput and parallelization of experiments. Each culturing unit in the array may still maintain an identifiable coordinate or index position within the overall system, which may be stored in the system’s control software to define the spatial relationship between the robotic arm 112 and the array positions.

[0053] In some embodiments, the coupling features 199 A and 199B may also include electrical or data interfaces, such as spring-loaded contacts or magnetic pogo pins, which automatically engage when the culturing unit 120 is attached. This allows each unit to receive power and communicate with the system controller without requiring separate cabling. The combination of mechanical, magnetic, and electrical coupling features enables rapid assembly, reconfiguration, or replacement of culturing units, enhancing the modularity and serviceability of the overall platform.

[0054] FIG. IE depicts another example implementation of the robotic platform system. This example system has been integrated into an anaerobic controlled atmosphere chamber, enabling microbes to be cultured under such conditions.

[0055] The culturing unit 120 is configured to monitor growth of a microbial population in the culture tube 122 (e.g., a 10-30 mL volume culture tube). Moreover, the culture unit 120 is configured to control culture temperature, aeration, and / or mixing using integrated heating elements, magnet-based stirrers, and / or the like. To monitor growth or dose cultures, the culture tube (which is inserted within the culturing unit 120) is illuminated by light sources positioned at multiple possible angles relative to a photodiode, allowing determination of culture density through absorbance and scattering measurements. As noted, a plurality of culturing tubes may eachDocket No. 24636-778 WO 1 be inserted into a corresponding culturing unit in the culturing system depicted at FIG. 1C. The culturing unit’ s heating element allow temperature control over the ambient temperature, and the culturing module’s magnet-based stirrers enable mixing and aeration (as depicted at Fig. 2B).

[0056] FIG. 2 A depicts an example perspective view of the culturing unit 120 having an opening 202 at a top surface 204. The opening 202 may have a similar shape and size as the culturing tube 122 (e.g., circular) such that the culturing tube 122 can be inserted into the opening. The opening 202 of the culturing unit may have a chamfered edge to assist the robotic arm in inserting or removing the tubes. Each culture module may contain a single culture tube at a time, which can be easily added and removed through the opening 202. The culturing tube 122 may have a friction or sliding fit with the opening 202. A friction or sliding fit may provide a seal between the culturing tube and the opening to aid in facilitating temperature control. The opening 202 may have a compliant inner circumference 203. The compliant material may be an elastomer, such as a thermoplastic elastomer. In some embodiments, the compliant material may be silicone rubber, thermoplastic polyurethane, EPDM rubber, O-rings, or similar materials.

[0057] FIG. 2B and 2C depict cut-away and exploded views of the culturing unit 120. In the example of FIG. 2B, the culturing unit 120 includes a magnetic stirring fan 230 to mix and aerate the culture tube 122. The magnetic stirring fan is within the culturing unit 120 and below a bottom interior portion 232 of the culturing unit 120. The interior cavity 237 (into which the culture tube 122 is inserted) is attached to the casing of the unit via a screwing mechanism to allow the cavity to be changed for the accommodation of different culture tube dimensions. Moreover, the culturing unit 120 may include a heating element 235. The heating element 235 may be comprised as a flexible PCB that wraps around the interior cavity 237 of the culturing unit 120. The flexible PCB 235 that wraps around interior cavity 237 may also contain light sources, which may beDocket No. 24636-778 WO 1 aligned with apertures 231 into the interior cavity 244 to illuminate the culture tube 122. The heater may be used to for example warm the culture above the ambient temperature.

[0058] As noted, the culturing unit 120 may include one or more sensors 231 and / or light sources 233 (e.g., photodiode, LEDs, temperature sensor, pH sensor, dissolved oxygen sensor, stirring sensor). These may be used to monitor the growth in the culture tube by for example measuring absorbance and scattering of light through the contained culture tube, with multiple selectable wavelengths. They may also be used to monitor culture conditions or dose cultures with light, by for example measuring temperature, measuring dissolved oxygen, measuring stirring rate, measuring pH, and / or activating light at multiple, selectable wavelengths.

[0059] In some embodiments, the culturing unit 120 may incorporate a suite of optical, environmental, and mechanical sensors configured to provide real-time monitoring and control of culture conditions during adaptive laboratory evolution experiments. The sensors may be positioned in predetermined locations within the culturing unit to obtain accurate and repeatable measurements without interfering with robotic access to the culture tube. The sensors may be operatively coupled to the unit’s primary printed circuit board (PCB) for continuous data acquisition and may communicate with the system controller to provide closed-loop control of growth parameters such as temperature, mixing rate, and aeration.

[0060] Optical sensors may include one or more photodiodes, phototransistors, or photometric detector arrays positioned to detect transmitted and / or scattered light through the culture tube. Light-emitting diodes (LEDs) may be located on opposing or adjacent sides of the tube cavity, arranged such that one or more detectors receive light at different angles relative to the illumination axis, for example at 90 degrees and 180 degrees. Measurements obtained from these optical paths can be used to determine optical density (OD), scattering intensity, andDocket No. 24636-778 WO 1 absorbance spectra of the culture, which in turn correspond to microbial concentration and growth rate. Multiple LEDs emitting at different wavelengths (for example, 450 nm, 600 nm, 800 nm, and 940 nm) may be employed to capture wavelength-dependent absorption profiles that distinguish between different cell states or culture media compositions. The LEDs and photodiodes may be mounted on flexible or rigid circuit boards that wrap around the culture tube cavity to ensure uniform illumination and detection geometry across all units.

[0061] Temperature sensors may include thermistors, resistance temperature detectors (RTDs), or solid-state temperature transducers embedded in the culturing unit wall or in thermal contact with the outer surface of the culture tube. These sensors provide real-time feedback on the temperature of the culture environment, allowing the control software to regulate the heating element 235 to maintain target temperatures or induce programmed thermal variations as part of selection pressure protocols. In some implementations, redundant temperature sensors may be positioned at different heights along the tube cavity to monitor thermal uniformity and detect potential stratification effects in the culture fluid.

[0062] To assess oxygenation and gas exchange conditions, the culturing unit 120 may include one or more dissolved oxygen (DO) sensors. These may be optical fluorescence-based sensors or electrochemical Clark-type sensors located in contact with the outer wall of the culture tube or integrated into the culture tube cap. The DO sensors provide measurements of oxygen concentration in the culture media, which may be used to infer metabolic activity and to dynamically adjust stirring rates or aeration conditions. Similarly, pH sensors, which may be optical dye-based or solid-state ion-sensitive field effect transistor (ISFET) sensors, may be incorporated in contact regions adjacent to the culture tube wall to continuously monitor changes in acidity during microbial growth.Docket No. 24636-778 WO 1

[0063] Mechanical or dynamic sensors may also be integrated into the culturing unit 120 to monitor physical parameters associated with mixing and agitation. For example, a magnetic stirring sensor may include a Hall effect sensor or magnetoresistive element positioned below the magnetic stirring fan 230 to detect its rotational speed. This allows verification that the stirring mechanism is operating as commanded and provides real-time data for feedback control of mixing rates. Vibration or accelerometer sensors may also be included to detect abnormal mechanical conditions such as imbalanced stirring, excessive vibration, or blockage within the culture tube.

[0064] In some embodiments, the sensors within the culturing unit 120 are arranged in a modular configuration that allows for expansion or reconfiguration depending on the experimental requirements. For example, optional sensor ports may be provided on the unit housing to accept plug-in sensor modules for specialized measurements, such as optical fluorescence emission, turbidity at non-standard wavelengths, or electrical conductivity. Each sensor module may be automatically recognized by the control firmware through a digital identification interface, enabling rapid configuration changes without hardware modification.

[0065] The spatial arrangement of the sensors within the culturing unit 120 may be designed to maintain consistent geometric relationships relative to the culture tube. The optical sensors may be symmetrically distributed around the tube cavity to ensure uniform detection, while environmental sensors such as temperature and pH sensors may be positioned along the lower portion of the cavity. The placement of these sensors in known, fixed positions enables the process control software to accurately interpret sensor signals and correlate them with physical parameters of the culture environment. The arrangement also facilitates calibration across multiple culturing units, ensuring consistent measurements between experiments and across batches.Docket No. 24636-778 WO 1

[0066] Referring to FIG. 2C and 2D, active elements of the culturing unit 120 may be controlled by a primary unit PCB 240 (FIG. 2B) using an integrated microcontroller 282, which contains a USB-C connector 284 for easy firmware updates, although other types of processors may be used to provide the noted control. Up to 8 culture units can be attached to a base plate PCBs 124, and up to 8 base plate PCBs can be simultaneously connected to one control unit 286. The control unit 286 has a microcontroller 282, similar to the one in the culture unit 120, which also contains a USB-C connector 284 for easy firmware updates and communication with a control PC 289. The base plate PCBs provides power and data connection to each culture unit, and the control unit provides power and data connection to each base plate.

[0067] Culture tubes may be inserted and removed by either operators or robot arms, allowing the contained microbial populations to be propagated to fresh media when appropriate. Culture tube lids 167 may be removed while in the culture units, allowing for supplements to be added during microbial growth or for aliquots to be removed for use in other equipment. Although this example refers to 8 base plates and a single controller, other quantities may be implemented as well.

[0068] FIG. 3A depicts aspects of the robotic platform. The disclosed platform may be arranged in a wide variety of configurations and installed in many different enclosures, FIG. 3A depicts an example configuration of components. For example, FIG. 3A depicts a system 300, which may have some or all of the components of system 100, that includes t the robotic arm 112, media dispensing station 350, a waste bin 312, a micropipette tip box 314, culture system 110, culturing units 120, culture tube storage racks 316, an end effector 320 which may include tube manipulation figures and micropipettor, coupled to a base, such as surface 102.Docket No. 24636-778 WO 1

[0069] In some embodiments, the robotic platform system 300 includes a robotic arm 112, which may be a multi-axis manipulator configured to transfer culture tubes, pipette tips, and other laboratory consumables between the various components of the system. The robotic arm 112 may be mounted to a fixed base or the surface 102 and may have sufficient reach and degrees of freedom to access all active elements of the platform. The arm may operate under programmatic control by the process control software, enabling automated workflows such as media dispensing, propagation, and sampling.

[0070] The media dispensing station 350, described in more detail below, may include one or more syringe pumps, valve manifolds, and fluid lines configured to dispense growth media and supplements into culture tubes. Each pump may be associated with a specific media component, allowing for dynamic formulation of growth media compositions during culturing. The dispensing station 350 may include one or more nozzle outlets or ports positioned at fixed coordinates accessible by the robotic arm 112 for accurate media delivery into open culture tubes.

[0071] The waste bin 312 may be configured to receive used consumables, such as discarded culture tubes, pipette tips, or other disposable materials generated during operation. The waste bin may be removable for cleaning or replacement and may include sensors to detect fill level or confirm that the bin is properly seated before operation. In some implementations, the waste bin 312 may also include a containment lid or filter element to prevent contamination or evaporation.

[0072] The micropipette tip box 314 may store disposable pipette tips compatible with the micropipettor of the end effector 320. The box may be located at a known position on the surface 102 so that the robotic arm 112 can automatically attach and eject tips during liquid handling operations. The box may include multiple tip trays arranged in a linear or stackedDocket No. 24636-778 WO 1 configuration, and may optionally include sensors or fiducial markings to confirm tip presence and alignment.

[0073] The culture system 110 may include one or more culturing units 120 arranged in a linear or array configuration as described herein. Each culturing unit may be configured to maintain microbial cultures under controlled temperature, stirring, and illumination conditions, and to provide real-time monitoring of growth parameters via integrated sensors. The robotic arm 112 may access each culturing unit for operations such as inoculation, propagation, and sampling.

[0074] The culture tube storage racks 316 may be configured to hold concentrated media stock components, unused or completed culture tubes in indexed positions that are accessible to the robotic arm 112. Each position may be uniquely identified within the system’s coordinate framework, allowing the control software to track tube status, location, and experimental association. The racks may be removable or replaceable to facilitate batch processing and sample storage.

[0075] In some embodiments, the robotic platform system may include devices to prepare samples for analytical analysis. Fig. IF depicts an embodiment that contains a positive pressure filtration device 128 mounted to the surface 102. Periodically, the platform may utilize the micropipettor attachment on the robot arm 112 to transfer aliquots of culture from the culture system 110 to a filter plate 130. Positive pressure removes cells as the culture passes through the filter, and the resulting filtrate may be analyzed using methods that may include high-performance liquid chromatography, liquid chromatography-mass spectrophotometry, or the like.

[0076] The end effector 320 coupled to the distal end of the robotic arm 112 may include one or more functional tools, such as tube manipulation fingers (e.g., a gripper) and a micropipettor. The gripper may be configured to grasp, lift, and transport culture tubes betweenDocket No. 24636-778 WO 1 the various system components, while the micropipettor may be used for aspirating and dispensing small liquid volumes. The end effector may be detachable or interchangeable to enable different tool configurations depending on the operational requirements of the experiment.

[0077] The entire system 300 may be mounted on or referenced to a base or surface 102, which defines the platform’s spatial coordinate system. The positions of the functional components — such as the culturing system 110, storage racks 316, media dispenser 350, and waste bin 312 — may be predetermined relative to the surface 102, allowing the robotic arm 112 to perform automated operations with precise positional repeatability.

[0078] FIG. 3B illustrates the robot’s arm with a gripper module 320 containing tube manipulation fingers that are configured to move culture tubes between the culture units, culture tube storage, and growth media dispensing station. Attached to the gripper module is a micropipettor 340 that enables both the propagation of experiments from one batch to the next and the addition of growth media components less than 1 milliliter from supplement tube storage to culture tubes. The gripper module may also include a tool adapter 338 for simultaneously coupling the micro-pipettor 340 and the tube manipulation fingers to the robotic arm 118.

[0079] FIG. 3C and 3D depict an example of a growth media dispensing station 350 using syringe pumps 356 to add growth media components greater than 1 milliliter to culture tubes. The multiple syringe pumps 356 and supplement tube or storage rack positions enable growth media to be dynamically formulated during culturing. The dispensing station 350 may include a housing on which one or more syringe pumps 356 are mounted. The housing 352 may enclose a volume for routing tubing to and from the syringe pumps. In some embodiments, growth media may be located within the housing. In some embodiments, growth media may be located externalDocket No. 24636-778 WO 1 to the housing. The growth media may be dispensed via one or more dispensing tips, each tip being coupled in fluid communication with a respective one of the syringe pumps.

[0080] FIG. 3D depicts the flow of culture grown media from a container 360 through a syringe pump 356, out a dispensing tip 358, and into a culture tube 122, which may then be placed into a culturing unit 120.

[0081] Platform hardware may be utilized to, for example, store reagents and supplies and to propagate the cultures. Fabricated racks enable storage of culture tubes and pipette tips. The growth media dispensing station utilizes syringe pumps to fill culture tubes with bulk growth media components from large storage bottles (FIG. 3C), and supplement tube storage positions to add small volumes of more concentrated growth media components. The growth media components can be added in any order and in any volume, enabling the dynamic creation of different growth medias during operation. The robot arm uses a gripper module with purpose-built tube manipulation fingers to move culture tubes between culture tube storage racks, culture units, growth media dispensing stations, and other, user-specified, satellite equipment. An adapter 338 attaches a micropipette to the gripper module (FIG. 3B), enabling the addition of small volumes of concentrated growth media components (e.g„ antibiotics) to culture tubes and the propagation of experiments to new culture tubes. The platform is largely layout agnostic, enabling installation in a wide variety of footprints and specialized environment enclosures. Example layouts are disclosed (FIGs. 3A, 1C, and IE).

[0082] In some implementations, a Python-based process control software package processes and stores data, generates a schedule for when various actions should occur, and communicates with the system hardware (FIG. 4), although other types of control may be implemented as well. Illustrative actions that the software may schedule include initial experimentDocket No. 24636-778 WO 1 batch inoculation, final experiment batch discarding, culture density measurement, and experiment propagation, the last of which may also include changes in selection pressures (Table 1, FIG. 5). Multiple selection pressure packages may be used to develop different traits in the experiments. The basic ALE selection pressure package maintains the microbial populations in an exponential growth phase under consistent or predetermined, sequentially alternating conditions, represented by FIG. 6A. A tolerization selection pressure package dynamically adjusts the culture temperature, stirring, or growth media composition between batches to incrementally increase stress to a tolerable level, as depicted by FIG. 6B. And. a non-native condition selection pressure package facilitates growth on new compounds by gradually reducing a growth-enabling supplement between batches until growth is observed in a “test” batch without the supplement, as represented by FIG. 6C.

[0083] Table 1 depicts anoverview of actions performed by the ALEpy process control software. The actions in Table 1 encompass actions that the platform performs as part of adaptive laboratory evolution or culturing experiments. The process control software may use the selection pressure package (FIG. 6) stored in each experiment (FIG. 4) to determine which actions to perform and at what time. Diagrams of how these actions are performed are included below (FIG. 5)

[0084] Table 1Docket No. 24636-778 WO 1

[0085] FIG. 4A depicts an overview of process control software and data structure used 400 by the platform comprising the culturing modules. The process, which in the embodiment herein are implemented by control software in cooperation with the hardware discussed herein, stores each run in a controller class instance (see, e.g., FIG. 4A), which contains machine, experiment, and growth media class instances. The controller class also has a function that generates a list of actions (Table 1 and FIGs. 5A-C) that the system uses to perform each experiment and the timing of each action. The machine class (see, e.g., FIG. 4B) stores information used to operate the platform, including communication settings for the culture units, micropipettor, collaborative robot arm, and syringe pumps, along with information on what is currently being stored in each culture unit position, culture tube storage position, and micro-pipettor tip box position. The experiment class (see, e.g., FIG. 4C) stores information on each experiment’s selection pressure, as well as each batch with its corresponding measurement types and measurements. Growth media classes store information on the growth media component composition of each growth media.Docket No. 24636-778 WO 1

[0086] FIGs. 5A-5C depict examples of workflows for the platform comprising the culturing modules platform operation. The overall process is described in FIG. 5A. When the process control software first starts in step 502B, the software may use inputs specified by the user in step 502A to create controller (FIG. 4A) and machine (FIG. 4B) instances. The controller instance created in step 502B may then have newly created experiment instances (Fig. 4C) added to it in step 502C, using parameters specified by the user in step 502A. The software may then inoculate (Table 1) each experiment instance in step 502D. The process control software then generates a schedule of actions that may be performed for each experiment in step 502E by inputting the current data for each experiment instance (FIG. 4C) into the corresponding selection pressure package instance (FIG. 6A) and then executing the chronologically soonest action at the scheduled time in step 502F. Steps 502E and 502F are then performed in an indefinite loop. After any execution of the generation of the schedule of actions in step 502E, the user may optionally intervene in step 502G to stop some (or all) of the experiment instances. If individual experiments are stopped, the control software may perform the discard action (Table 1) to stop that experiment.

[0087] Examples of possible actions selected in step 502E are shown in Tables 1 and FIGs. 5B and 5C. The measure action (see, e.g., 5B) collects sensor information, which in the example shown here includes for example culture density, temperature, and stirring rate, from a culture unit and stores it in the batch instance currently associated with that unit. Once the software has begun the measure action in step 504A, the stirring rate of the culture unit being measured may be reduced in step 504B, if specified in the selection pressure package for the associated experiment instance (FIG. 6A). The temperature value is then read in 504C. and the culture absorbance or scattering or other light-based sensor measurements are determined, for example, in triplicate by flashing the specified light source(s) in step 504D and reading the photodiodeDocket No. 24636-778 WO 1 value(s) in step 504E while the light is emitted. In some embodiments, such as under fluorescence measurements, the sensor or photo diode may measure light after the light emitter is turned off. In some Embodiments, the stirring rate of the culture unit being measured is then increased back to the starting value in step 504F, and the software calculates the culture density in step 504G using an average of the readings from the photodiode. These values are then added to a new measurement instance and measurement type instance in the batch class instance of the experiment instance (FIG. 4C) in step 504H, and the measure action ends.

[0088] The propagate action (FIG. 5C-1 and FIG. 5C-2) transfers an aliquot of liquid from a culture tube on a culture unit to a new culture tube, creating a new batch instance for the related experiment instance. This enables the microbial population to continue growing and, depending on the selection pressure package for that experiment, may adjust culture conditions such as growth media composition, temperature, or aeration. When the propagate action first begins in step 506A, the data for the current batch instance of the experiment instance may be analyzed in the relevant selection pressure package instance (FIG. 4C) in step 506B to determine if propagation should occur, if not the action may be terminated at this point. As part of step 506B, a measure action may be performed in step 506C. Once it is determined that propagation should commence in step 506B, the software may identify an unused culture tube on the storage rack in step 506D and remove the lid from this culture tube in 506F. The software may then fill this unused culture tube with growth media in steps 506G-506K. For each component of the growth media, the software may move the culture tube to the component location in 506G. For media components that are attached to the media dispensing station (FIG. 3C), the media component may be directly dispensed by the syringe pump (part of 116) in step 506E. For each component that is stored in a supplement tube, the software may first remove the supplement tube lid (step 506H), attach aDocket No. 24636-778 WO 1 micropipettor tip to the micropipettor 118 on the robotic arm 112 (step 5061), use the micropipettor 118 to transfer the aliquot of the component (step 506J), dispose of the tip, and replace the supplement tube lid in step 506K. Once the desired media components have been added to the unused tube, the tube may then be moved temporarily to an unused culturing unit in step 506L and optionally be heated. The lids may be removed from the current batch culture tube in step 506M, and a micropipettor tip may be added to the micropipettor 118 on the robotic arm 112 in step 506N. The micropipettor 118 may then be used to transfer the aliquot of the culture from the current batch culture tube to the unused culture tube in step 5060. and in step 506P the micropipettor tip may be disposed of and lids placed back on both culture tubes. The current culture tube is then removed from its culturing unit and placed in an empty culture tube storage rack position in step 506Q. In step 506R, the unused culture tube is moved to the culturing unit assigned to this experiment instance, and the culturing unit stirring is reset and a measurement action performed in step 506S. And, a new batch instance is created for this culture tube and added to the experiment instance associated with this culturing unit in step 506T, completing the propagate action.

[0089] FIGS. 6A-C illustrate an example of selection pressure packages of the software. In the basic ALE selection pressure package (FIG. 6A), microbial populations can be maintained in constant or fixed sequence alternating growth media compositions, stirring rates, and temperatures. Fixed-rate culture density sampling is used, and experiment propagation occurs when a user-specified culture density is reached. A tolerization selection pressure package (FIG. 6B) dynamically adjusts the culture temperature, stirring, or growth media composition between batches to dynamically adjust stress levels. Stress is increased if a maximum growth rate is reached and decreased if a minimum growth rate is not reached. And, a non-native condition selection pressure package facilitates growth on new compounds by gradually reducing a growth-enablingDocket No. 24636-778 WO 1 supplement between batches until a minimum allowable culture density is reached. Simultaneously, it may inoculate “test” batches without the growth-enabling supplement, and once these begin to grow the growth-enabling supplement microbial population is terminated (FIG. 6C).

[0090] The platform comprising the culturing modules described herein may provide one or more advantages. For example, the overall platform is designed to be extremely scalable, and any number of each of the components (robotic arms, culturing units, dispensing station syringe pumps, etc.) can be easily integrated to scale throughput up or down. Larger systems require only minimally more labor hours from operators than the smaller systems. Moreover, the platform layout can be redesigned very flexibly depending on available space, as evidenced by FIGs 1C and IE. In addition to the illustrative layout described herein, other embodiments have been developed which fit inside an anaerobic chamber (FIG. IE) and yet other embodiments have also been developed with a larger footprint. Related to this, the platform can easily integrate with other analytical equipment because the cultures are accessible by the micropipettor and robotic arm during operation, enabling aliquots to be transferred to the analytical equipment without interrupting the system or requiring operator interaction Further, the selection pressure packages available in the process control software are able to develop specific desired traits in the cultures, such as tolerance to stressful compounds or growth on compounds that normally do not support it. And, the dispensing unit having multiple dispensing lines and syringe pumps, and the micropipettor being able to access growth media components on the supplement tube storage positions, enable media to be formulated differently from batch to batch and change over the course of operation.

[0091] In some embodiments, FIG. 7A depicts an assembled culturing unit 120 including a housing 128 that encloses internal mechanical, thermal, optical, and electronic subsystems. AnDocket No. 24636-778 WO 1 aperture 202 in an upper portion of the housing 128 provides access for a culture tube 122 to be removably received within a tube cavity of the unit. The tube cavity geometry may be sized to accept a target tube diameter and length, and in some embodiments may be exchanged or adjusted to accommodate different tube formats as described herein.

[0092] FIG. 7B illustrates the culturing unit 120 with the outer housing 128 removed to reveal an internal housing structure 129 mounted on abase 241. The internal housing structure 129 supports the relative positions of the primary unit PCB 240, a magnetic stirring fan 230, a heater module with heating elements 325. and vertically arranged sensor PCBs. The base 241 provides a rigid datum for positional repeatability supports the stacked components described below. This vertically stacked arrangement of components includes integrated heating, magnet-based stirring, and optical sensing to control culture temperature, mixing, and optical density measurements.

[0093] In the illustrated embodiment, the primary unit PCB 240 is mounted directly to the base 241. The primary unit PCB 240 hosts power distribution, sensor interfaces, and control electronics, and may include a microcontroller 282 that communicates with a control unit and a control PC. A service connector, which in some embodiments is a USB-C connector 284, may provide firmware update and communications capability. The primary unit PCB 240 may also route signals to the sensor PCBs and to the heater module and stirring systems so that temperature, optical, mixing, and other signals are centrally managed.

[0094] Stacked above the primary unit PCB 240 is the magnetic stirring fan 230. The stirring fan 230 is positioned below the lower region of the tube cavity and is configured to magnetically couple to a stirring element within the culture tube 122 to provide mixing and aeration. In some embodiments, a magnet housing having magnets is coupled to the stirring fan 230 to transfer stirring energy more efficiently to the internal stirring device while maintaining aDocket No. 24636-778 WO 1 low profile in the vertical stack. A speed sensing device, such as a Hall effect sensor on the primary unit PCB 240 or elsewhere, such as on the heating PCB or the sensor PCBs, may monitor the rotational speed of the magnetic stirring fan 230 to confirm mixing setpoints and to enable closed- loop control of stirring during measurement and propagation actions. This is consistent with prior descriptions of magnet-based stirring for culture mixing and aeration.

[0095] A heater module with heating elements 325 is located above the magnetic stirring fan 230. The heating elements 325 are arranged so that, when a culture tube 122 is present, a lower portion of the tube is in close thermal proximity to the heater module to enable controlled heating of the culture fluid. The heater module is operatively controlled by the primary unit PCB 240 which reads temperature sensors and adjusts heater drive to maintain desired culture temperatures. Prior text describes the use of flexible PCB heaters around the interior cavity and temperature control of the culture, and the FIG. 7 arrangement provides an alternative stack that maintains those functional relationships while simplifying assembly.

[0096] The sensor PCBs may be arranged to extend generally vertically from the plane of the heater module and adjacent to the tube cavity. In one implementation, a light emitter PCB carries light emitters 233 that illuminate the culture tube 122 at selected wavelengths. A complementary light sensor PCB carries light sensors 231, which may include one or more photodiodes, that receive transmitted and / or scattered light. The sensor PCBs may be positioned such that optical paths at different angles, for example about 180 degrees for transmission and about 90 degrees for side scattering, can be obtained in a repeatable geometiy. The sensor PCBs may also carry environmental or dynamic sensors or portions of such sensors (such as connectors to read the sensors), such as temperature sensors, pH sensors, dissolved oxygen sensors, andDocket No. 24636-778 WO 1 stirring sensors as discussed herein. The vertical arrangement provides a compact optical baseline while maintaining clear access to the aperture 202 for robotic or operator tube handling.

[0097] In some embodiments, one or more of the printed circuit boards (PCBs) within the culturing unit 120 may be interchangeable or swappable to enable modular upgrades and configuration flexibility. For example, the sensor PCBs may be configured as removable modules that can be exchanged to provide different sensor packages or combinations of sensing capabilities. Each sensor PCB may contain a digital identifier, such as an EEPROM or embedded microcontroller, that communicates with the controller PCB 240 when the unit is powered on. This allows the system controller to automatically detect the specific sensors present and configure corresponding measurement routines within the control software. The software may also verify that the installed sensors support the measurements for a given process or experimental protocol. If a requested measurement cannot be performed, such as, for example, if the process control sequence calls for monitoring stirring speed but the stirring speed sensor is missing or nonfunctional, the controller may generate an error or warning, log the event, and optionally adjust the protocol to proceed safely with available sensors.

[0098] Similarly, other PCBs within the culturing unit 120 may be interchangeable to support hardware variation and scalability. The heater PCB may be swapped to provide different heating element geometries or densities, such as heating element arrangements optimized for particular tube diameters or volumes. In some embodiments, the heater modules may be heat transfer modules, that provide for the transfer of heat energy into or out of the tube in the culturing unit 120. For example, the heater module may be a Peltier module, water bath or tubes, or other heat transfer device. This modularity allows the same culturing unit architecture to accommodate multiple vessel sizes while maintaining efficient heat transfer. The primary or controller PCB 240Docket No. 24636-778 WO 1 may likewise be replaceable to enable hardware upgrades or functional customization, such as additional sensor interfaces, advanced communications hardware, or revised control algorithms.

[0099] FIG. 7C shows an exploded view in which the base 241, primary unit PCB 240, magnetic stirring fan 230, heater module with heating elements 325, and the sensor PCBs are aligned along a common reference axis established by the internal housing structure 129. Mechanical or electro-mechanical alignment features, such as pin headers, on the internal housing structure 129 may locate the internal elements in the lateral and vertical positions of each assembly maintain the optical emitters and sensors in fixed offsets from the tube cavity, allow the heater module to maintain consistent thermal contact with the tube 122, and that the stirring fan 230 remains coaxial with the stirring element within the culture tube 122. The resulting assembly allows repeatable measurement of culture density through absorbance and scattering as described previously, and operation of the heater and stirrer under the control of the primary unit PCB 240.

[0100] FIG. 7D provides cutaway views that illustrate component relationships. In some embodiments, the FIG. 7 architecture is implemented as a modular unit that mounts to a base plate or culturing system frame. Electrical and data connections from the unit may be made through edge connectors or cabling to a base plate PCB and to a control unit that aggregates data from multiple culturing units. The control unit communicates with a control PC that executes process control software for measuring, inoculating, propagating, and discarding batches as previously detailed. This is consistent with the multi-unit base plate arrangement and controller communications described for other figures.

[0101] In operation, the FIG. 7 embodiment supports the same measurement and action sequences discussed herein. The optical subsystem captures absorbance and scattering data at selected wavelengths, the heater module maintains temperature setpoints during growth or appliesDocket No. 24636-778 WO 1 temperature programs as part of selection pressure packages, and the stirring fan provides controlled mixing rates that may be temporarily reduced during measurement and then restored. These actions integrate with the machine and experiment data structures and with the selection pressure packages that define when to measure or propagate. In some embodiments, FIG. 8 depicts a system diagram of a system 800 that represents a variation of the system 200 previously described with respect to FIG. 2D. The system 800 includes separate hubs for data and power management to improve modularity and scalability of the platform. In this embodiment, a data hub 810 and a power hub 820 are provided. The data hub 810 may be a USB hub. Ethernet hub, or another digital communication hub configured to aggregate data signals from a plurality of base plates or culturing systems 124. The power hub 820 may be configured to distribute electrical power to the various components of the platform, including each culturing unit 120 or base plate 124. While the power lines between the power hub 820 and the individual culture plates 124 are not explicitly shown, it will be understood that the VCC and GND connections from the power hub 820 are electrically coupled to the corresponding VCC and GND lines of each base plate, thereby providing a common power supply reference across all connected units.

[0102] The separation of data and power hubs allows for differential scaling of power capacity and data throughput, which may be advantageous for installations involving large arrays of culturing units or high-frequency sensor sampling. For example, a single data hub 810 may support a greater number of culturing units when wireless data transfer is employed, while additional power hubs 820 may be added to accommodate expanded thermal or stirring requirements.

[0103] In other embodiments, one or more of the data connections between the base plates 124 and the data hub 810 may be implemented wirelessly. Each base plate 124 or culturingDocket No. 24636-778 WO 1 unit 120 may include a communications module configured to provide wireless data transfer. The communications module may be a Wi-Fi, Bluetooth, Zigbee, or other short-range wireless transceiver that communicates either with the data hub 810 or directly with the central control system 289. Wireless communication allows flexible system layouts and reduces cable congestion, which can be beneficial when the system is installed in an environmental enclosure or anaerobic chamber. Each wireless-enabled unit may have a unique identifier that allows the control software to associate sensor readings and actuator commands with the correct physical unit. In some versions, hybrid communication may be used, in which certain signals, such as time-critical actuator commands, are transmitted through wired connections, while sensor data and status updates are transmitted wirelessly.

[0104] In some implementations, data may also be transferred wirelessly between individual culturing units 120 and either the data hub 810 or the control system 289. This configuration may be particularly useful for distributed or modular installations where culturing units are physically separated or mounted in different arrays, hi some configurations, each culturing unit may include an onboard microcontroller that buffers and timestamps local sensor data before transmission, ensuring synchronization of measurements across multiple units.

[0105] In some embodiments, the system 800 may also include redundant data paths or backup communication channels. For example, if wireless communication between a culturing unit 120 and the data hub 810 is interrupted, the system may automatically reroute communication through a wired connection or a secondary wireless interface. Likewise, the power hub 820 may incorporate a redundant power supply or backup battery system to maintain culture viability during short-term power interruptions. This level of redundancy allows the system 800 to maintain continuous operation in laboratory environments where reliability and data integrity are critical.Docket No. 24636-778 WO 1

[0106] The above examples and disclosure are intended to be illustrative and not exhaustive. These examples and description will suggest many variations and alternatives to one of ordinary skill in this art. All these alternatives and variations are intended to be included within the scope of the attached claims. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the claims attached hereto.

Claims

Docket No. 24636-778 WO 1CLAIMSWhat is claimed:

1. A robotic culturing system, comprising: a plurality of culturing units; a robotic arm configured to transfer culture tubes between the plurality of culturing units, a culture tube storage rack, and a media dispensing station; and a processor and memory storing instructions that, when executed by the processor, cause the system to carry out a method comprising: measuring, by one or more sensors of at least one of the plurality of culturing units, parameters of a culture within a tube; transmitting sensor data from the culturing unit to the processor; analyzing, by the processor, the sensor data to determine a growth status of a microbial population within one of the plurality of culturing units; determining, based on the analyzed growth status, whether propagation of the microbial population should occur; in response to determining that propagation should occur: controlling the robotic arm to receive growth media into an empty culture tube; controlling the robotic arm to transfer an aliquot of the microbial population from a current culture tube to the new culture tube; and initiating a new batch of microbial culture in the new culture tube.

2. The system of claim 1, wherein determining whether propagation should occur comprises comparing the measured culture density to a predefined propagation threshold corresponding to an exponential growth phase of the microbial population.

3. The system of claim 1, further comprising, in in response to determining that propagation should occur controlling the media dispensing station to dispense growth media into the empty culture tube;Docket No. 24636-778 WO 14. The system of claim 3, wherein controlling the media dispensing station comprises actuating a plurality of syringe pumps, each associated with a distinct media component, to formulate a growth media mixture according to a selection pressure package.

5. The system of claim 1, wherein the method further comprises adjusting at least one of temperature, stirring rate, or media composition between successive batches to implement an adaptive laboratory evolution selection pressure.

6. The system of claim 1, wherein the robotic arm comprises an end effector having both a gripper and a micropipettor, and the step of transferring the aliquot comprises: using the gripper to remove lids from the culture tubes; attaching a pipette tip to the micropipettor; aspirating the aliquot from the current culture tube and dispensing it into the new culture tube; and discarding the used pipette tip after the transfer.

7. The system of claim 1, wherein the method further comprises replacing the culture tube in the culturing unit with the newly inoculated tube and recording the association between the new tube and an updated batch identifier in the control software.

8. The system of claim 1, further comprising generating, by the processor, a schedule of actions comprising a sequence of measuring actions and propagation actions for each experiment and executing the sequence based on the current state of each culture tube.

9. The system of claim 1, further comprising calculating, from repeated measuring actions, a growth rate or fitness parameter for each microbial population and adjusting future propagation timing based on the growth rate or fitness parameter.

10. The system of claim 1, wherein measuring the parameters of the culture tube comprises:Docket No. 24636-778 WO 1 illuminating the culture tube using one or more light emitters disposed on a first sensor PCB; and detecting light transmitted through or scattered from the culture tube using one or more photodiodes disposed on a second sensor PCB positioned at approximately 90 degrees or 180 degrees relative to the light emitters.

11. The system of claim 1, further comprising adjusting a stirring speed of the culture tube during the measuring action, wherein the stirring speed is temporarily reduced to minimize optical disturbance and subsequently restored after the measurement.

12. The system of claim 1, further wherein each of the plurality of culturing units includes: a cavity configured to receive a culture tube; one or more light sources arranged to direct light through the culture tube; one or more light sensors positioned to detect light transmitted through or scattered by the culture tube; at least one heating element configured to control temperature of the culture tube; a magnetic stirring arrangement positioned beneath the cavity and configured to mix and aerate contents of the culture tube; and a primary printed circuit board (PCB) in operative communication with the one or more light sensors, the one or more light sources, the heating element, and the magnetic stirring arrangement.

13. The system of claim 12, wherein the heater module has one or more heating elements arranged adjacent to a bottom portion of the culture tube to provide bottom-up heating.

14. The system of claim 1, wherein each of the culturing units includes at least one coupling feature configured to removably attach the culturing unit to a base plate.Docket No. 24636-778 WO 115. The system of claim 13, wherein the coupling feature comprises a snap-fit mechanism, a magnetic coupling, or an alignment pin or keyed channel configured to maintain a fixed spatial position relative to a platform coordinate system.

16. The system of claim 1, wherein the plurality of culturing units are arranged in one of: a linear configuration, in which the culturing units are positioned along a single axis; or a two-dimensional array configuration, in which the culturing units are positioned in rows and columns to form a grid.

17. The system of claim 1, wherein the robotic arm includes an end effector comprising: a gripper configured to grasp and move culture tubes; and a micropipettor configured to aspirate and dispense small liquid volumes between culture tubes or storage containers.

18. A culturing method, the method comprising: measuring, by one or more sensors of at least one of a plurality of culturing units, parameters of a culture within a tube; transmitting sensor data from the culturing unit to a processor; analyzing, by the processor, the sensor data to determine a growth status of a microbial population within one of the plurality of culturing units; determining, based on the analyzed growth status, whether propagation of the microbial population should occur; in response to determining that propagation should occur: controlling a media dispensing station to dispense growth media into an empty culture tube; controlling a robotic arm to transfer an aliquot of the microbial population from a current culture tube to the new culture tube; and initiating a new batch of microbial culture in the new culture tube.Docket No. 24636-778 WO 119. The method of claim 18, wherein in response to determining that propagation should occur, controlling a robotic arm to dispense small volumes of concentrated media components into the fresh media tube;20. The method of claim 18, wherein determining whether propagation should occur comprises comparing the measured culture density to a predefined propagation threshold corresponding to an exponential growth phase of the microbial population.

21. The method of claim 18, further comprising adjusting at least one of temperature, stirring rate, or media composition between successive batches to implement an adaptive laboratory evolution selection pressure.

22. The method of claim 18, wherein transferring the aliquot comprises: using the gripper to remove lids from the culture tubes; attaching a pipette tip to the micropipettor; aspirating the aliquot from the current culture tube and dispensing it into the new culture tube; and discarding the used pipette tip after the transfer.

23. The method of claim 18, further comprising replacing the culture tube in the culturing unit with the newly inoculated tube and recording the association between the new tube and an updated batch identifier in the control software.

24. The method of claim 18, further comprising generating, by the processor, a schedule of actions comprising a sequence of measuring actions and propagation actions for each experiment and executing the sequence based on the current state of each culture tube.

25. A culturing apparatus, comprising: a cavity configured to at least partial contain a culture tube;Docket No. 24636-778 WO 1 at least one light source disposed around a tube holder element to direct light through the culture tube; a photodiode arrangement disposed around at least a portion of the tube holder element to receive light transmitted through the culture tube for performing absorbance and / or scattering measurements; at least one heating element surrounding at least a portion of the tube holder element; a magnetic-based stirring arrangement disposed on a bottom portion of the culturing apparatus for mixing and aerating a solution located in a culture tube inserted in the tube holder element; and a primary printed circuit board (PCB) unit in operative communication with the photodiode arrangement, the at least one light source, the at least one heating elements, and the magnetic-based stirring arrangement.

26. The culturing apparatus of claim 1 wherein the culture tube has a removable lid that is removable while culturing of microbial populations is in progress.

27. A platform for culturing microbes and / or performing adaptive laboratory evolution, comprising: a plurality of culturing apparatus as set forth in claim 1 ; a robot arm having a gripper module at its distal end; a culture tube storage arrangement; a media dispensing unit having a plurality of dispensing lines and syringe pumps for dispensing growth media into the culture tubes, the gripper module being configured to insert the culture tubes into and remove the culture tubes from the tube holder elements in the plurality of culturing apparatus and transfer culture tubes to and from the culture tube storage arrangement and the media dispensing unit, the gripper module including a micropipettor for injection of liquids into the culture tubes and transfer of microbial population aliquots from one culture tube to another culture tube; at least one base plate arrangement having a plurality of base plates, each adapted to removably receive at least one of the plurality of culturing apparatus, so that the base plates are in electrically operable communication with, and supplies power to, the respective at least one of the plurality of culturing apparatus; andDocket No. 24636-778 WO 1 at least one processor to control actions performed by the robot arm and the plurality of culturing apparatus.

28. The platform of claim 3 wherein the processor is further in operable communication with the syringe pumps and the micropipettor.

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