GB2618589A

GB2618589A - Bioreactor system

Info

Publication number: GB2618589A
Application number: GB2206912.4A
Authority: GB
Inventors: Seng Wong Tuck; Joseph Rady Brooks; Lan Tee Kang
Original assignee: University of Sheffield
Current assignee: University of Sheffield
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-11-15
Also published as: WO2023218166A1

Abstract

A bioreactor system for cultivating a pool of cells using adaptive laboratory evolution, the bioreactor system comprising: a cultivation and diversification module 100 for cultivating a pool of cells; a selection module 20 configured to apply a selective pressure (e.g. chemical mutagen or mutagenic virus) to the pool of cells during cultivation; a sensor module 300 in fluid communication with the cultivation and diversification module and configured to measure data indicative of a condition of the pool of cells, and a control module operatively connected to the selection module and the sensor module and configured to operate the selection module to adjust the selective pressure based on the measured data, wherein the sensor module is separate from the cultivation and diversification module. The cultivation and diversification module may also comprise an ionizing radiation source in the lid assembly 110 emitting e.g. UV light, X-rays or gamma rays to illuminate the cells and thereby resulting in mutagenesis.

Description

BIOREACTOR SYSTEM

TECHNICAL FIELD

[0001] The present invention relates to a bioreactor system and particularly, but not exclusively, to use of a bioreactor system, for strain optimisation, strain characterization, scouting of growth conditions/selective conditions of cells, and understanding the mechanisms of evolution. Aspects of the invention relate to a bioreactor system and use of a bioreactor system.

BACKGROUND

[0002] Adaptive laboratory evolution (ALE) is an emerging tool for biological discovery and industrial biotechnology. It provides molecular insights into the mechanism of evolution, and a means to adapt cell properties to suit industrial applications (e.g., biomanufacturing). Using ALE, cell factories can be adapted to biomanufacturing conditions, resulting in higher production yield and lowering production cost. Conventionally, ALE is conducted using either: serial cultivation or chemostat. In the case of serial cultivation, batch cultivation is performed in shake flasks to propagate cells, and at regular time intervals, an aliquot of the culture is transferred to a new flask with fresh medium for the next round of growth. This process is iterated until optimised cells are obtained. In the case of chemostat, cells are continuously cultured in a bioreactor for a prolonged period of time. Regardless of the chosen approach (serial cultivation or chemostat), all successful evolution campaigns include two key elements: (a) genetic diversity, and (b) selective pressure.

[0003] Bioreactors are vessels or tanks in which whole cells or cell-free enzymes transform raw materials into biochemical products. Existing bioreactors are mostly designed for fermentation, cell cultivation, biotransformation, and water treatment. They are not designed for strain optimisation, strain characterization, scouting of growth conditions/selective conditions of cells, and understanding the mechanism of evolution. They lack either or both of the capabilities of creating genetic diversity and applying selective pressure. Existing bioreactors also have limited ability to switch between cultivation modes. The vessel size, the sensor probes, and the control software are not suitable for strain optimisation. Although ALE can be performed in flasks or culture tubes, doing so requires extensive manual handling. Not only is the procedure labour intensive, it is prone to human error and contamination. Importantly, there is often no real-time or continuous cell optical density measurement in flask and tube cultivation, making it difficult to perform ALE systematically.

[0004] It is an aim of certain examples of the present invention to solve, mitigate or obviate, at least partly, at least one of the problems and/or disadvantages associated with the prior art. Certain examples aim to provide at least one of the advantages described below.

BRIEF SUMMARY OF THE INVENTION

[0005] According to an aspect of the invention, there is provided a bioreactor system for cultivating a pool of cells using adaptive laboratory evolution, the bioreactor system comprising: a cultivation and diversification module for cultivating a pool of cells; a selection module configured to apply a selective pressure to the pool of cells during cultivation; a sensor module in fluid communication with the cultivation and diversification module for and configured to measure data indicative of a condition of the pool of cells, and a control module operatively connected to the selection module and the sensor module and configured to operate the selection module to adjust the selective pressure based on the measured data. The sensor module is separate from the cultivation and diversification module.

[0006] The present bioreactor system advantageously provides a flexible and intelligent bioreactor system for adaptive laboratory evolution to optimise the performance of prokaryotic or eukaryotic cells and to understand the evolution of viruses. The present modular design also allows for the addition or removal of one or more modules depending on specific application.

[0007] The bioreactor system may comprise a mixing unit arranged to mix the pool of cells within the cultivation and diversification module. The mixing unit may be arranged to rotate a stirring element in the cultivation and diversification module. The mixing unit may be arranged to rotate the stirring element in a bidirectional manner to improve mixing and gas-liquid mass transfer. The stirring element may comprise a magnetic element and/or an overhead stirrer. The mixing unit may comprise a Hall sensor for detecting the rotation of the magnetic element.

[0008] The cultivation and diversification module may comprise one or more ports for any of the following: inoculating the pool of cells, sampling, modifying and/or draining the cultivation and diversification module. The cultivation and diversification module may comprise a reactor vessel and a lid assembly. The lid assembly may comprise the one or more ports. The cultivation and diversification module may comprise one or more sampling lines extending from the one or more ports into the pool of cells. Each of the one or more sampling lines may be a resilient tube. The resilient tube may comprise metal, such as stainless steel, glass or polymeric materials.

[0009] The control module may be operatively connected to the cultivation and diversification module. The bioreactor system, preferably the cultivation and diversification module, may comprise a temperature control unit configured to heat and/or cool the cultivation and diversification module, preferably the reactor vessel. The temperature control unit may comprise a thermal transfer plate having a first portion arranged under the cultivation and diversification module for heating and/or cooling the cultivation and diversification module. The first portion may comprise a plurality of segments extending under the cultivation and diversification module from a common end section. Each segment may comprise a first end and a second connected to the common end section. The temperature control unit may comprise a peltier unit or a heating/cooling element operatively coupled to the thermal transfer plate. In some cases, the temperature control unit can be part of the selection module.

[0010] The sensor module may be arranged to measure an optical property of the pool of cells. The optical property may be any one or more of an optical density, absorbance, turbidity and fluorescence. The sensor module may comprise a pump arranged to circulate a sample of the pool of cells between the sensor module and the cultivation and diversification module.

[0011] The cultivation and diversification module may comprise an ionizing radiation source. Suitably, the ionizing radiation source may emit a UV light, an x-ray, and/or a gamma ray beam. The ionizing radiation source may be arranged to illuminate the pool of cells. Illuminating the pool of cells with ionizing radiation (such as UV light, X-ray, and/or gamma ray beams) may result in mutagenesis of some or all of the cells in the pool of cells. The UV light source may be a UVA, UVB or UVC light source or any combination of these light sources. The UV light source may comprise a plurality of UV LEDs. The UV light source may be arranged along the circumference of the reactor vessel or beneath the lid assembly. The selection module may be configured to introduce a chemical mutagen and/or a mutagenic virus into the cultivation and diversification module. Merely by way of example, the chemical mutagen may be selected from the group consisting of hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea, Nethyl-N-nitrosourea (EN U), 0-methyl hydroxylamine, nitrous acid, methyl methane sulfonate (MMS), ethyl methane sulfonate (EMS), sodium bisulfite, formic acid, Mil'', and nucleotide analogues.

[0012] The bioreactor system may comprise a liquid level sensor arranged to detect a liquid level within the cultivation and diversification module. The liquid level sensor may be integral to the reactor vessel or the lid assembly.

[0013] The control module may be configured to output the measured data to a remote device. The control module may be configured to determine the condition of the pool of cells based on the measured data. The control module may be configured to receive a signal indicative of the condition of the pool of cells from the remote device, and configured to control the selection module and/or the cultivation and diversification module based on the signal received from the remote device. The control module may be configured to connect to a wide area network, such as the Internet. The control module may comprise a microcontroller, a Raspberry Pi, or combination of these. The control module may be configured to receive a control signal from a remote device for operating the bioreactor assembly. The control module may be configured to operate any of: the selection module and the cultivation and diversification module based on the received control signal. The control module may be configured to perform any of: collection, visualisation, and analysis of the data from the sensor module.

[0014] The selection module may comprise at least one pump arranged to modify the growth medium composition within the cultivation and diversification module. The cultivation and diversification module may be a first of a plurality of cultivation and diversification modules of the bioreactor system. The first cultivation module may be fluidly connected to a second cultivation and diversification module of the plurality of cultivation and diversification modules. The selection module may be configured to pump an aliquot of cultivated cells from the first cultivation and diversification module to the second cultivation and diversification module. It would be apparent that a plurality of cultivation and diversification modules could be connected in this way, for example in series (where one cultivation and diversification module is connected to only one other cultivation and diversification module) and/or in a parallel arrangement (where one cultivation and diversification module is connected to multiple cultivation and diversification modules). The control module may be configured to operate the selection module to pump the aliquot of cultivated cells to the second cultivation and diversification module based on the detected condition reaching a pre-determined threshold. The selection module may comprise one or more DC motor-driven pumps and/or stepper motor-driven pumps, such as a peristaltic pump, for transferring liquid to and/or from the cultivation and diversification module. The selection module may comprise a temperature control unit arranged to heat and/or cool the pool of cells within the cultivation and diversification module.

[0015] The control module may be configured to: determine the condition of the pool of cells based on one or more of: determining an optical property measured by the sensor module is greater than a first user-defined variable, and/or determining if the pool of cells have gained fitness is true, and/or determining if a time of cultivation is greater than a second user-defined variable, and/or determining if a specific growth rate measured by the sensor module approaching a third user-defined variable is true, and/or determining if an inflection point of a growth curve measured by the sensor module has passed is true.

[0016] There is also provided a processor comprising a non-volatile memory with instructions stored thereon for operating the control module as described herein.

[0017] There is also provided a use of a bioreactor system as defined herein for identifying selective pressure conditions capable of producing and/or enriching a cellular sub-population, for producing a cell with a desired phenotype, and/or enriching a cellular sub-population with a desired phenotype, for strain optimisation, strain characterization, scouting of growth conditions/selective conditions of cells, and/or studying the mechanisms of evolution.

[0018] In another aspect, the present invention provides a method of producing a cell sub-population having a desired phenotype, the method comprising: a) providing a pool of cells to a cultivation and diversification module of the bioreactor of the present invention; and b) applying a selective pressure to enrich for cells having the desired phenotype, optionally wherein prior to step b) the pool of cells undergoes the step of mutagenesis.

[0019] Provided is also use of a bioreactor system of the present invention in adaptive laboratory evolution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Examples of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 is a perspective illustration of an exemplary bioreactor system; Figure 2 is a perspective illustration of an exemplary cultivation and diversification module and sensor module; Figure 3 is a cross-sectional view of the modules of Figure 2; Figure 4 is a perspective illustration of an exemplary pump; Figure 5 is a cross-sectional side view of an exemplary sensor module; Figure 6 is a perspective illustration of an exemplary thermal transfer plate, and Figure 7 is an alternative cross-sectional view of the modules of Figure 2. DETAILED DESCRIPTION [0021] Figure 1 illustrates an exemplary bioreactor system 10. The illustrated bioreactor system 10 includes a selection module 20, a cultivation and diversification module 100 having a temperature control unit 400, a mixing unit 200 and a sensor module 300 for monitoring a condition of a cellular pool within the cultivation and diversification module 100 as is explained below. While the illustrated bioreactor system 10 includes a mixing unit 200 it would be apparent this is not essential. The sensor module 300 is also shown separate to the reactor vessel 105, which is advantageous over reactor vessels 105 with an integrated sensor module. Having an independent (i.e. separated) sensor module 300 from the cultivation and diversification module 100 provides a modular bioreactor system 10 with multiple advantages over the prior art. For example, having a separate sensor module 300 means the bioreactor system 10 is not limited by the size of the reactor vessel 105, as a larger reactor vessel 105 results in lower sensitivity when used with an integrated sensor (i.e. a sensor permanently attached to the reactor vessel 105). A further advantage of separating the sensor module 300 is that the present bioreactor system 10 has a much larger dynamic range and sensitivity because the light path through a sample 307 within the sensor module 300 (see Figure 5) is better controlled compared to measurements within the main reactor vessel in prior art systems. A further advantage of a separate sensor module 300 is that the bioreactor system 10 can be customised more easily depending on the particular optical property being measured. Thus, an appropriate sensor module 300 can be selected for a given application (e.g., an absorbance-only sensor module, a fluorescence-only sensor module, or an absorbance and fluorescence sensor module). The present selection module 20 includes pumps 25 for media exchange or for changing media composition of the cultivation and diversification module 100. In some cases, the selection module 20 can introduce one or more inhibitors or mutagens (such as chemical mutagens and/or mutagenic viruses) into the reactor vessel 105. As such, the present bioreactor system 10 can be used in various cultivation modes such as batch cultivation, continuous cultivation (e.g., chemostat and turbidostat), and semi-continuous cultivation (e.g., serial cultivation). Semi-continuous cultivation is particularly advantageous in adaptive laboratory evolution (ALE) studies, but is not essential.

[0022] The cultivation and diversification module 100 is mounted on a support platform 15 and includes a reactor vessel 105 for cellular cultivation, and a lid assembly 110 secured to the top of the reactor vessel 105. The reactor vessel 105 may have any cross-sectional area and shape, but in the illustrated bioreactor 10, the reactor vessel 105 is a cylindrical vessel. This is advantageous as a cylindrical vessel resembles how cells are cultivated in a large-scale vessel in industrial processes. This is particularly advantageous over systems where cultivation takes place in a tube, which: suffer from potential biofilm formation within the tube (which can escape the selective pressure), require a large foot print to perform, are difficult to scale and do not reflect industrial bioprocesses.

[0023] With reference to Figures 2 to 3, the lid assembly 110 includes multiple (six are shown) ports 115, 120 for delivering and removing liquid from within the reactor vessel 105. The ports 115, 120 are in fluid communication with a respective liquid source or drainage source via the selection module 20 which, in the illustrated example, includes four pumps 25. The sensor module 300 is also fluidly connected to a pump 25A which enables circulation of a sample 307 between the cultivation and diversification module 100 to and the sensor module 300 as explained below. The illustrated pump 25 is a peristaltic pump (see Figure 4) which has a rotor 30 for driving liquid through a tube (not shown) extending from an inlet 27 to an outlet 29 of the pump 25. The pump 25 is preferably driven by a stepper motor which is advantageous over a DC motor-driven pump, as this enables multiple pumps to be synchronised with each other such that the volume of liquid entering the reactor vessel 105 is equal to the volume of liquid being removed from the reactor vessel 105. This is an important element of various cellular cultivation modes. In contrast, DC motor-driven pumps may operate at different speeds due to internal friction even if driven at the same voltage. As a consequence, the liquid level within the reactor vessel 105 may change.

[0024] As shown in Figure 3, the lid assembly 110 has a recess 130 for a sensor (not shown) to monitor the conditions within the reactor vessel 105. In some cases the sensor is a liquid level sensor, which advantageously allows for monitoring of the liquid level within the reactor vessel 105.

[0025] As illustrated, ports 115A, 115B are for introducing liquid 107 into the reactor vessel 105 and ports 120A, 1203 are for draining liquid 107 from the reactor vessel 105. Ports 120A, 1203 are shown as barbed members having a respective sampling line 125A, 125B extending therefrom towards the base of the reactor vessel 105 such that the sampling lines 125A, 125B extend close to the base. As shown in Figure 3, the sampling lines 125A, 125B are straight and, when the reactor vessel 105 contains liquid 107, such as the cellular pool and any growth medium, the sampling lines 125A, 125B are at least partially submerged in the liquid 107. This allows port 120A to be used for draining while port 120B is used to circulate culture through the sensor module 300. By way of example, stainless steel sampling lines 125A, 125B advantageously act as baffles within the liquid which facilitates mixing within the liquid 107. However, it would be apparent that a sampling line 125 made of stainless steel is merely an example of a resilient material which would stiffen the sampling line 125 sufficiently to act as a baffle in the liquid 107. Other metals or polymer or glass materials would be suitable for this function.

[0026] The mixing unit 200 illustrated in Figure 3 is housed within the platform 15 underneath the reactor vessel 105. The mixing unit 200 includes a motor 205 for rotating a stirrer 210 having a magnetic element 215. The stirrer 210 is arranged in close proximity to a stirring element 220 contained within the reactor vessel 105. As the stirring element 220 has a corresponding magnetic element 225, rotation of the stirrer 210 causes a corresponding rotation of the stirring element 220 which facilitates mixing of the liquid 107 within the reactor vessel 105. The stirring element 220 is sized to fit between the ends of the sampling lines 125A, 125B to avoid interference between the stirring element 220 and the sampling lines 125A, 125B. The mixing unit 200 also includes a mount 230 for a Hall sensor 235. Providing the Hall sensor 235 allows for precise measurement of the rotation speed of the stirrer 210, and therefore the rotation speed of the stirring element 220. As the viscosity of the liquid 107 changes, the rotation speed can therefore be adjusted accordingly to a greater degree of accuracy. The motor 205 can also be selectively driven to rotate in two directions. Bi-directional rotation advantageously provides better mixing of the liquid 107 compared to stirrers that rotate in one direction only. A controller (not shown) can be used to automatically adjust the rotation speed of the stirrer and/or the frequency with which the rotation direction changes. It would be apparent that this could be in addition or as an alternative to manual user inputs to adjust the rotation speed or rotation direction.

[0027] Figure 2 shows the sensor module 300 mounted to the platform 15 independently of (i.e. physically separated from) the reactor vessel 105. The sensor module 300 is connected to the reactor vessel 105 by a liquid line (not shown) so that a sample 307 of the liquid 107 within the reactor vessel 105 can be inspected. The sample 307 can be inspected according to a number of conditions to estimate the condition of the liquid 107 within the reaction vessel 105. In one example, an optical condition, such as optical density, of the sample 307 may be measured via the sensor module 300.

[0028] As shown in Figure 5, the sensor module 300 has a housing 305, an inlet port 310, an outlet port 315 and a fluid flow path extending between the inlet portion 310 and the outlet port 315. The housing 305 acts as a light shield for the optical measurements (e.g. by comprising an optically opaque material) and has a further viewing port 325 for viewing a portion of the fluid flow path within the housing 305. An optically transparent section 320 is provided within the housing 305 so that the optical density of the sample 307 taken from the reactor vessel 105 can be inspected. As the optical density reaches a pre-determined threshold, this can be used to adjust the selective pressure applied to the liquid 107 within the reactor vessel 105. The separate sensor module 300 provides: higher sensitivity, especially for ALE, where the reactor vessel 105 has a volume of approximately 100 mL; greater sensing flexibility as sensor modules 300 can be added or removed as required; volume flexibility as different sized reactor vessels 105 can be used without compromising sensing sensitivity in some cases. Optical density is one example of an optical property suitable for determining the condition of the sample 307, but it would be apparent that the condition of the sample 307 could be measured according to other optical properties, such as absorbance and/or fluorescence measurements. In combination with the liquid level sensor data, it is possible to accurately measure the liquid volume within the reactor vessel 105, which in turn can be used to calculate the starting optical density value for cellular cultivation.

[0029] With reference to Figures 2, 3, 6 and 7, the temperature control unit 400 is mounted to the platform 15 and is arranged to provide the required cultivation temperature to influence the evolution of the cellular pool within the reactor vessel 105. In the illustrated example, the temperature control unit 400 includes a mount 405 for a pelfier unit (not shown) for heating/cooling the reactor vessel 105 to evolve thermotolerance and cold adaptation. A thermally conductive plate 410 is connected to the peltier unit and/or mount 405 to adjust the temperature within the reactor vessel 105. As shown in Figures 3, 6 and 7, the thermally conductive plate 410 is an L-shaped plate having a vertical section 420 connected to a horizontal section 415 which extends underneath the reaction vessel 105 and is in contact with the reaction vessel 105. The horizontal section 415 includes a comb-shaped heat transfer element formed of five separate elements 430A-430E. However, it would be apparent fewer or more than five separate elements 430A-430E could be used depending on the requirements of the temperature control unit 400. Each heat transfer element 430A-430E extends from a first end to a second end, both of which are connected to a connection section 425 which connects the horizontal section 415 and vertical section 420. The transfer elements 430A-430E have a linear portion adjacent each end, and a circular portion. The circular portions of the heat transfer elements 430A-430E are preferably concentric with one another. This advantageously breaks the eddy current caused by the magnetic stirrer 210 positioned below the heat transfer elements 430A-430E. It would be appreciated that the illustrated thermally conductive plate 410 is merely one example of a thermal conductor suitable for heating/cooling the reactor vessel 105, and that the illustrated thermally conductive plate 410 is not essential to the bioreactor system. It would also be apparent that other heating and/or cooling components could be included in addition to or alternatively to a pelfier unit to provide heating and or cooling in the temperature control unit 400.

[0030] The bioreactor system 10, specifically the cultivation and diversification module 100 includes multiple UVC LEDs for controlling the mutation rate. These can be arranged within the cultivation and diversification module 100, for example around a circumference of the lid assembly 110. The UV light source is one way to provide the diversification of the pool of cells within the reactor vessel 105. It would be apparent other means, such as other ionizing radiation sources, chemical mutagens, and/or mutagenic viruses would also be suitable to provide diversification of the pool of cells.

[0031] The bioreactor system 10 is operatively connected to a remote device to enable remote control of the bioreactor system 10 and its constituent parts to perform the functions described above. The bioreactor system 10 preferably includes a microcontroller to perform the functions described above. This advantageously consumes less power compared to traditional personal computers. In fact, the autonomous operation provided by the microcontroller means the bioreactor system 10 does not require a separate local control unit or computer to operate. The present bioreactor system 10 can therefore be scaled up more easily compared to traditional systems and can be used in parallel with multiple similar devices. The microcontroller is operatively connected to a communication unit for establishing a network connection, such as a Wide Area Network (WAN) connection, for remote control. Where the WAN connection is the Internet, the present bioreactor system 10 can be controlled remotely via a webpage independent of a particular computer operating system, which further increases the flexibility of the bioreactor system 10. The microcontroller can have a non-volatile memory for storing instructions for implementing the desired cultivation mode. Additionally or alternatively, a remote device may contain the instructions for implementing the desired cultivation mode and the microcontroller may simply execute the received instructions. The remote device may have a user interface for receiving a user input for executing some or all of the instructions for the bioreactor system 10.

[0032] An exemplary method for performing adaptive laboratory evolution is provided below: Step 1: Fill the bioreactor vessel with a mL of culture medium, where a is a user-defined variable, for example between 5 and 80mL, preferably 50mL. The medium may or may not contain inhibitor(s), for example inhibitors "A" and/or "B" described below.

Step 2: Inoculating the bioreactor vessel with b pt of overnight culture, where b is a user-defined variable, for example between 5 and 2000 pL, preferably 500 pL.

Step 3: Cell growth at any of: a user-defined temperature (e.g. between 5 and 50°C, preferably 37°C), a user-defined stirring speed (e.g. between 200 and 6000 revolutions per minute, preferably 500 revolutions per minute), and a user-defined stirring direction (e.g. clockwise when viewing the reactor vessel 105 from a plan perspective).

Step 4: Decision making by determining whether a condition of the pool of cells has reached a pre-determined condition.

Step 5: Remove d mL of culture, where d is a user-defined variable, for example between 4 and 79mL, preferably 48 mL Step 6: Add e mL of fresh medium, where e is a user-defined variable, for example between 4 and 79mL, preferably 48 mL.

Step 7: Go to Step 3 [0033] The decision making step may comprise any combination of a. determining if the optical density measured by the sensor module > c is true, where c is a user-defined variable (for example between 0.1 and 10, preferably 2.5), and/or b. determining if the cells that have gained fitness (i.e ability to grow under selective pressure) is true, and/or c. determining if a time of cultivation > c is true, where c is a user-defined variable (for example between 4 and 168 hours, preferably 24 hours), and/or d. determining if the specific growth rate measured by the sensor module approaching c is true, where c is a user-defined variable (for example between 0.01 to 1 h-1, preferably 0.15h-1), and/or e. determining if the inflection point of the growth curve measured by the sensor module has passed is true.

[0034] The method can include a step of mutagenesis. The step of mutagenesis can happen before or after cell growth. The step of mutagenesis can happen before or after the decision making step. Where there are multiple decision making steps, the step of mutagenesis can happen between decision making steps.

[0035] The method can be repeated from any of: the cell growth step or the mutagenesis step. It will be appreciated that mutagenesis may be UV mutagenesis, or any other type of mutagenesis known to the skilled person, such as other ionizing radiation, chemical, and/or viral mutagenesis.

[0036] The method can include adding one or more inhibitors "A" and "B", where A and B are user-defined inhibitors. The inhibitor(s) can be added after the step of adding fresh medium. By way of example, "A" and "B" can be chemicals in the lignocellulosic hydrolysate known as inhibit microbial growth such as phenolics (e.g., coumaric acid, syringaldehyde, vanillin), aliphatic acids (e.g., acetic acid, formic acid, levulinic acid), and furans (e.g., furfural, hydroxymethylfurfural).

[0037] It will be appreciated that examples of the present invention can be realized in the form of hardware, software or a combination of hardware and software. Any such software may be stored in the form of volatile or non-volatile storage, for example a storage device like a ROM, whether erasable or rewritable or not, or in the form of memory, for example RAM, memory chips, device or integrated circuits or on an optically or magnetically readable medium, for example a CD, DVD, magnetic disk or magnetic tape or the like. It will be appreciated that the storage devices and storage media are examples of machine-readable storage that are suitable for storing a program or programs comprising instructions that, when executed, implement examples of the present invention.

[0038] Accordingly, examples provide a program comprising code for implementing apparatus or a method as claimed in any one of the claims of this specification and a machine-readable storage storing such a program. Still further, such programs may be conveyed electronically via any medium, for example a communication signal carried over a wired or wireless connection and examples suitably encompass the same.

[0039] It will be appreciated from the following description that, in certain examples of the invention, features concerning the graphic design of user interfaces are combined with interaction steps or means to achieve a technical effect. It will be appreciated from the following description that, in certain examples of the invention, graphic features concerning technical information (e.g. internal machine states) are utilised to achieve a technical effect. Certain examples aim to achieve the technical effect of enhancing the precision of an input device. Certain examples aim to achieve the technical effect of lowering a burden (e.g. a cognitive, operative, operational, operating, or manipulative burden) of a user when performing certain computer or device interactions. Certain examples aim to achieve the technical effect of providing a more efficient man-machine (user-machine) interface.

[0040] In one aspect, the present invention provides use of a bioreactor system of the present invention for identifying selective pressure conditions capable of producing and/or enriching a cellular sub-population, for producing a cell with a desired phenotype, and/or enriching a cellular sub-population with a desired phenotype, for strain optimisation, strain characterization, scouting of growth conditions and/or selective conditions for producing and/or enriching cell, and/or studying the mechanisms of evolution.

[0041] It will be appreciated that the desired phenotype may be as a result of a specific (desired) genotype. The genotype that results in the desired phenotype may be achieved by subjecting a pool of cells to mutagenesis, for example random mutagenesis.

Mutagenesis may result in a mutation that gives rise to the desired phenotype. Such a mutation may be in a coding and/or non-coding region of the DNA. Methods of performing mutagenesis (for example random mutagenesis) will be known to those skilled in the art. Exemplary methods for performing mutagenesis are described elsewhere in the present description, and may include illuminating cells with an ionizing radiation source, and/or contacting cells with chemical mutagens and/or mutagenic viruses. Upon subjecting the pool of cells to mutagenesis (for example random mutagenesis), the cells may be exposed to selective pressure conditions in order to identify a sub-population of cells capable of being cultivated (i.e. surviving and/or growing in) under those pressure conditions. Suitably by cultivating cells under selective pressure a cell sub-population with a desired phenotype may be enriched. Merely by way of example, the desired phenotype may be specific substrate utilization, pH tolerance, thermotolerance, salt tolerance, inhibitor tolerance, solvent tolerance, increased productivity, increased yield, and/or faster growth. It will be appreciated that in the context of the present disclosure the term "phenotype" refers to a transient or persistent phenotype.

[0042] As used herein, the term "producing" refers to generating a cell that has a desired phenotype. The term "enriching" as used herein refers to the process of increasing the percentage of a specific cell that has a desired phenotype as compared to its percentage in the pool of cells prior to subjecting the cells to selective pressure. Suitably, selective pressure provides conditions that promote the growth of the specific sub-population of cells (such as cells with a desired phenotype) over other cells (such as cells that may have been initially present in the pool of cells prior to the application of selective pressure).

[0043] In the context of the present invention, the pool of cells may be subjected to mutagenesis (for example random mutagenesis) and selective conditions whilst in the reactor vessel (105). In such an embodiment, it can be said that the bioreactor is used for generating and enriching of a cell sub-population. Alternatively, the pool of cells may be subjected to mutagenesis (for example random mutagenesis) prior to providing the pool of cells in the reactor vessel (105). In other words, mutagenesis may be performed whilst the pool of cells is not in the reactor vessel (105). In such an embodiment, it can be said that the bioreactor is used for enriching a cellular sub-population. In some embodiments, the cells may be subjected to mutagenesis (for example random mutagenesis) before and whilst in the reactor vessel (105).

[0044] As used herein the term "strain optimisation" refers to producing and/or enriching a specific cell sub-population that has a desired phenotype. Such a cell sub-population may find utility in, for example, industrial biotechnology sector (bioenergy, biochemical, biomaterial, biopharmaceutical, and food sector). Suitably, strain optimisation may involve parallel cultivation of more than one cell sub-population, wherein each population is cultivated in a different reactor vessel (105), applying selective pressure conditions to each of the cell sub-population and selecting the sub-population that grows the best under said conditions. The phrase "scouting of growth conditions and/or selective conditions" refers to identifying different conditions which may result in the production and/or enrichment of a cell sub-population with a desired phenotype. Such conditions may be then applied outside of the bioreactor described herein to a pool of cells in order to produce and/or enrich a cell population having the desired phenotype.

[0045] The term "cell" as used herein means any eukaryotic or prokaryotic cell. The cell may be a mortal or immortalised cell. A eukaryotic cell refers to any animal, plant, yeast or fungal cell having a definitive nucleus. Eukaryotic cells of animals include cells of vertebrates, e.g., mammals, and cells of invertebrates, e.g., insects. In certain embodiments the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include but are not limited to mammalian cell lines. In one embodiment the mammalian cell is a human cell. Merely by way of example, the mammalian cell may be a HEK 293 cell, HeLa cell, a fibroblast, a Chinese hamster ovary (CHO) cell, and/or a baby hamster kidney (BHK) cell. Merely by way of example the yeast cell may be a Saccharomyces cerevisiae, KomagataeHa phaffii (formerly known as Pichia pastoris), Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, Rhodotorula toruloides, or Candida cell. Merely by way of example the fungal cell may be an Aspergillus, Fusarium, Penicillium, or Trichoderma cell.

[0046] A prokaryotic cell means any cell or any organism belonging to the Archaea or phylogenetic group of bacteria. The bacteria may be gram positive or gram negative. Examples of gram positive bacteria include Actinomyces, Bacillus, Lactobacillus, Carnobacterium, Leuconostoc, Oenococcus, Pediococcus, Tetragenococcus, Vagococcus, Weissella, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, and Mycobacterium. Examples of gram negative bacteria are Escherichia coli, Cupriavidus, Rhodococcus, Burkholderia, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter, Pseudomonas, Legionella, and Neisseria.

[0047] The phrase "a pool of cells" and/or "a cell sub-population" as used herein refers to a plurality of cells (i.e. 2 or more cells). The pool of cells and/or cell sub-population may be genetically homogenous (i.e. have substantially the same genotype) or be genetically heterogenous (i.e. have genetic differences, which can be natural genetic variations or men-made mutations, caused for example by mutagenesis as mentioned elsewhere herein).

[0048] In another aspect, the present invention provides a method of producing a cell sub-population having a desired phenotype, the method comprising: a) providing a pool of cells to a reactor vessel (105); and b) applying a selective pressure to enrich for cells having the desired phenotype Suitably, the method of producing a cell having a desired phenotype may comprise the step of subjecting the pool of cells to mutagenesis (for example random mutagenesis).

Suitably, the step of subjecting the pool of cells to mutagenesis may be carried out before or after step a). It will be appreciated that, suitably, the step of subjecting the pool of cells to mutagenesis may be carried out after step a) in an embodiment in which the bioreactor comprises means by which the cells may be mutated, such as a UV light source or other ionizing radiation source arranged to illuminate the pool of cells, and/or the selection module may be configured to introduce a chemical mutagen and/or a mutagenic virus into the cultivation and diversification module.

In a further aspect, provided herein is use of a bioreactor system of the present invention in adaptive laboratory evolution.

[0049] Throughout this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other components, integers or steps. Throughout this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Throughout this specification, the term "approximately" is used to provide flexibility to a range endpoint by providing that a given value may be "a little above" or "a little below" the endpoint. The degree of flexibility of this term can be dictated by the particular variable and can be determined based on experience and the associated description herein.

[0050] Features, integers or characteristics described in conjunction with a particular aspect or example of the invention are to be understood to be applicable to any other aspect or example described herein unless incompatible therewith. It will be appreciated that, throughout this specification, language in the general form of "X for Y" (where Y is some action, activity or step and X is some means for carrying out that action, activity or step) encompasses means X adapted or arranged specifically, but not exclusively, to do Y. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

Claims

CLAIMS: 1. A bioreactor system for cultivating a pool of cells using adaptive laboratory evolution, the bioreactor system comprising: a cultivation and diversification module for cultivating a pool of cells; a selection module configured to apply a selective pressure to the pool of cells during cultivation; a sensor module in fluid communication with the cultivation and diversification module and configured to measure data indicative of a condition of the pool of cells, and a control module operatively connected to the selection module and the sensor module and configured to operate the selection module to adjust the selective pressure based on the measured data, wherein the sensor module is separate from the cultivation and diversification module.
2. A bioreactor system according to claim 1, comprising a mixing unit arranged to mix the pool of cells within the cultivation and diversification module.
3. A bioreactor system according to claim 2, wherein the mixing unit is arranged to rotate a stirring element in the cultivation and diversification module.
4. A bioreactor system according to claim 2 or 3, wherein the stirring element comprises a magnetic element.
5. A bioreactor system according to claim 4, wherein the mixing unit comprises a Hall sensor for detecting the rotation of the magnetic element.
6. A bioreactor system according to any preceding claim, wherein the cultivation and diversification module comprises one or more ports for inoculating the pool of cells.
7. A bioreactor system according to claim 6, wherein the cultivation and diversification module comprises one or more sampling lines extending from the one or more ports into the pool of cells.
8. A bioreactor system according to claim 7, wherein each of the one or more sampling lines are a resilient tube.
9. A bioreactor system according to any preceding claim, wherein the cultivation and diversification module comprises a reactor vessel for cultivating the pool of cells, and wherein the bioreactor system comprises a temperature control unit configured to heat and/or cool the cultivation and diversification module.
10. A bioreactor system according to claim 9, wherein the temperature control unit comprises a thermal transfer plate having a first portion arranged under the cultivation and diversification module for heating and/or cooling the cultivation and diversification module.
11. A bioreactor system according to claim 10, wherein the first portion comprises a plurality of segments extending under the cultivation and diversification module from a common end section, and wherein each segment comprises a first end and a second connected to the common end section.
12. A bioreactor system according to any preceding claim, wherein the sensor module is arranged to measure an optical property of the pool of cells.
13. A bioreactor system according to any preceding claim, wherein the cultivation and diversification module comprises a UV light source arranged to illuminate the pool of cells.
14. A bioreactor system according to any preceding claim, wherein the bioreactor system comprises a liquid level sensor arranged to detect a liquid level within the cultivation and diversification module.
15. A bioreactor system according to claim 14 when dependent on claim 9, wherein the liquid level sensor is integral to the reactor vessel.
16. A bioreactor system according to any preceding claim, wherein the control module is configured to output the measured data to a remote device.
17. A bioreactor system according to any preceding claim, wherein the control module is configured to connect to a wide area network.
18. A bioreactor system according to any preceding claim, wherein the control module is configured to receive a control signal from a remote device, and wherein the control module is configured to operate any of: the selection module and the cultivation and diversification module based on the received control signal.
19. A bioreactor system according to any preceding claim, wherein the cultivation and diversification module is a first of a plurality of cultivation and diversification modules of the bioreactor system, and wherein the first cultivation module is fluidly connected to a second cultivation and diversification module of the plurality of cultivation and diversification modules, and wherein the selection module is configured to pump an aliquot of cultivated cells from the first cultivation and diversification module to the second cultivation and diversification module.
20. A bioreactor system according to any preceding claim, wherein the control module is configured to: determine the condition of the pool of cells based on one or more of: determining an optical property measured by the sensor module is greater than a first user-defined variable, and/or determining if the pool of cells have gained fitness is true, and/or determining if a time of cultivation is greater than a second user-defined variable, and/or determining if a specific growth rate measured by the sensor module approaching a third user-defined variable is true, and/or determining if an inflection point of a growth curve measured by the sensor module has passed is true.
21. A bioreactor system according to any preceding claim, wherein the selection module is configured to introduce an inhibitor into the cultivation and diversification 15 module.
22. A processor comprising a non-volatile memory with instructions stored thereon for operating the control module according to any preceding claim.
23. Use of a bioreactor system of any one of claims 1 to 21 for identifying selective pressure conditions capable of producing and/or enriching a cellular sub-population, for producing a cell with a desired phenotype, and/or for enriching a cellular sub-population with a desired phenotype, for strain optimisation, strain characterization, scouting of growth conditions/selective conditions of cells, and/or studying the mechanisms of evolution.
24. A method of producing a cell sub-population having a desired phenotype, the method comprising: a) providing a pool of cells to a cultivation and diversification module of the bioreactor system of any one of claim 1 to 21; and b) applying a selective pressure to enrich for cells having the desired phenotype.
25. Use of a bioreactor system of any one of claims 1 to 21 in adaptive laboratory evolution.