GB2563577A - Methods and apparatus for selection and characterisation of genetically transformed cells - Google Patents

Abstract

A genetic material testing system in which a sequence of droplets is formed.The droplet formation is controlled to form droplets that comprise, onaverage, no more than N biological cells per droplet wherein the ratio of un­transformed biological cells to transformed biological cells in the droplet is approximately N:1. Sample of cells from each colony grown in a droplet are deposited onto a substrate comprising a material that is capable of supportingcell growth, in an organised manner, to form a predetermined pattern ofsamples wherein each position of the predetermined pattern is traceable back to a corresponding colony from which the cells at that position originated.

Description

Methods and apparatus for selection and characterisation of genetically transformed cells

The present invention relates to methods for selection and for characterisation of transformed cells and to associated apparatus. The invention has particular although not exclusive relevance to selection and characterisation of genetic circuits or devices that have been inserted into transformed cells.

In molecular biology, ‘transformation’ is a process by which a cell is genetically altered as a result of the incorporation of exogenous genetic material through the cell’s membrane(s).

In Synthetic Biology, the process of transformation is used to insert newly designed genetic circuits, which are coded into DNA sequences, into cells with the objective of modifying the behaviour of the cells, for example to make them behave in a predetermined way. These genetic circuits are designed with a specific purpose in mind and have to be tested, or characterised, to ensure the transformed cell responds as intended. This characterisation usually involves exposing the transformed cells to one or more analytes and/or conditions that trigger a desired response from the genetic circuit.

Current characterisation techniques rely heavily on manual handling of samples, involving a number of steps and the use of large quantities of consumables. Cells are normally suspended in a liquid buffer solution. Analyte is mixed into the buffer solution in order to expose the cells to the analyte and, after an incubation period, the response of the cell is quantified, for example by means of an optical measurement system.

In order to characterise the full dynamic response of the cell, cells with the new genetic circuits, devices or the like are exposed to a range of concentrations of the analyte. Each response experiment is carried out on an independent group of cells (i.e. one group per analyte concentration). The response is measured at each analyte concentration of the range of concentrations with the results combined together to create a dynamic response profile. Ideally, one response profile is obtained for each genetic circuit being characterised. The genetic design with the performance that best matches the desired response can then be identified from these profiles.

In order to obtain a meaningful response profile it is typical to use several (typically between 8 to 12) different concentrations levels per analyte, per genetic design, meaning that the number of characterisation experiments can quickly escalate to significant numbers, particularly if analysis of a large number of genetic designs is required. This requires large equipment, for example with the ability to handle large number of microtiter plates or other such equipment, usually involving large amount of consumables.

In view of all these requirements, therefore, current characterisation processes can be very time and resource consuming, involving lengthy steps and requiring a lot of manual interventions. Moreover, manual characterisation processes such as these are particularly susceptible to human error. As a result, generally, only a handful of genetic designs can be characterised within a reasonable timescale and at a reasonable cost thus limiting the amount of information that can be made available to a researcher.

The proposed invention is aimed at addressing or at least partially ameliorating one or more of these issues. The invention seeks to provide a method and associated apparatus for contributing to achieving this aim.

In one aspect the invention provides apparatus, for a genetic material testing system, the apparatus comprising: means for receiving (e.g. an input of a microfluidic chip or the like) a mixture comprising un-transformed biological cells and transformed biological cells, wherein each transformed cell comprises at least one genetic design to be tested; and means for forming (e.g. a dropletising microfluidic chip or the like), from the mixture, a sequence of droplets, wherein the droplet formation is controlled to form droplets that comprise, on average, no more than N biological cells per droplet wherein the ratio of un-transformed biological cells to transformed biological cells in the droplet is approximately N:1.

The apparatus may comprise means for growing (e.g. an incubator or the like), in-situ in each droplet, a cell colony from any transformed biological cell in that droplet. Each transformed cell may comprise at least one selection marker and said means for growing (e.g. incubator or the like) is operable to select, based on the at least one selection marker and in-situ in each droplet, for any transformed biological cell in a particular droplet (and against un-transformed cells in that particular droplet).

The apparatus may comprise means for screening the sequence of droplets to sort droplets that do not comprise any transformed biological cells from droplets that do comprise at least one transformed biological cell or a colony grown from at least one transformed biological cell. For example, the means for screening may comprise a microfluidic device, or the like, with a complementary optical transmitter and optical detector arranged to pass light through a droplet for receipt by the receiver and a microfluidic switch arranged to switch the fluidic pathway along which the drops are passed in dependence on the turbidity of the droplet as indicated by the amount of light passing through it and detected by the detector. The means for screening the sequence of droplets may be further configured to sort droplets that comprise a monoclonal colony grown from a single transformed biological cell from droplets that comprise a polyclonal colony grown from a plurality of different transformed biological cells.

The means for screening the sequence of droplets may be operable to provide, to characterisation apparatus, a sorted sequence of droplets, wherein each of at least a majority of droplets (e.g. at least 50% or greater, but typically 75% or greater, or more preferably 90% or greater) in the sorted sequence comprises a single respective monoclonal colony grown from a single transformed biological cell.

In one aspect the invention provides characterisation apparatus, for a genetic material testing system, the apparatus comprising: means for receiving a sequence of droplets (e.g. a microfluidic conduit), wherein each of at least a majority of droplets in the sequence comprises a single respective monoclonal colony grown from a single transformed biological cell comprising at least one genetic design to be tested; means for depositing (e.g. at least one pin, or array of pins, arranged for pin-spotting of biological samples) at least one respective sample of cells from each colony onto a substrate comprising a material that is capable of supporting cell growth, in an organised manner, to form a predetermined pattern of samples wherein each position of the predetermined pattern is traceable back to a corresponding colony from which the cells at that position originated; and means for characterising a respective response of each deposited sample, in-situ on the substrate, to at least one external factor to which that sample is exposed. The means for characterising may, for example, comprise any suitable imaging device (such as a digital camera) with an associated imaging head and associated optics for capturing images. It may also comprise a computer arranged to analyse images from the imaging apparatus and to compare results of such analysis to a predetermined desired response.

The apparatus may comprise means for storing at least a sample of cells from each said colony in a liquid growth medium at a different respective known location, wherein each sample of cells from each colony is respectively taken from a corresponding known location for said depositing onto the substrate, and wherein each position of the predetermined pattern is traceable back to a corresponding known location of said means for storing. The means for storing may comprise, for example any suitable biological storage element such as a well plate, or the like. The samples may be taken using any appropriate tool such as a pipetting tool, or the like, that may be configured for taking multiple samples, substantially simultaneously from different locations (e.g. wells) of a biological storage element (e.g. well plate).

The means for depositing may be arranged to deposit samples from the same colony on each of a plurality of substrates for exposing the samples to a different respective external factor.

The response to at least one external factor may comprise a response to at least one analyte and wherein the analyte is integrated into the substrate, prior to deposition of the deposited samples. The means for depositing may be arranged to deposit respective samples from the same colony on each of a plurality of substrates and wherein each of the plurality of substrates comprises a different respective known concentration of the at least one analyte integrated into that substrate prior to deposition of the deposited samples.

The pattern may comprise an array having a plurality of columns and a plurality of rows. A plurality of samples from the same colony may be deposited at a plurality of different locations in the same column (or row) and a plurality of samples from different colonies are deposited at a plurality of different locations in the same row (or column).

Each transformed biological cell from which a colony is grown may comprise at least one reporting marker; and wherein the respective response of a deposited sample, to the presence of the external factor, arises from the presence of the at least one reporting marker in the transformed biological cell from which that deposited sample originated.

Each transformed biological cell from which one of the colonies is grown may comprise at least a first reporting marker and a second reporting marker; and the characterisation means may be configured to perform ratiometric characterisation of a deposited sample based on a ratio of a first response of that deposited sample arising from the first reporting marker to a second response of that deposited sample arising from the second reporting marker. The, or each, reporting marker may be configured to provide an optical response (e.g. a fluorescent or luminescent response) and the characterisation means may be configured to measure the optical response.

In one aspect the invention provides a genetic material testing system comprising, apparatus as recited in the first mentioned aspect and characterisation apparatus as recited in the second mentioned aspect.

In one aspect the invention provides a method performed in a genetic material testing system, the method comprising: receiving a mixture comprising un-transformed biological cells and transformed biological cells, wherein each transformed cell comprises at least one genetic design to be tested; and forming, from the mixture, a sequence of droplets, wherein the droplet formation is controlled to form droplets that comprise, on average, no more than N biological cells per droplet wherein the ratio of un-transformed biological cells to transformed biological cells in the droplet is approximately N:1.

In one aspect the invention provides a method performed by a genetic material testing system the method comprising: receiving a sequence of droplets, wherein each of at least a majority of droplets in the sequence comprises a single respective monoclonal colony grown from a single transformed biological cell comprising at least one genetic design to be tested; depositing at least one respective sample of cells from each colony onto a substrate comprising a material that is capable of supporting cell growth, in an organised manner, to form a predetermined pattern of samples wherein each position of the predetermined pattern is traceable back to a corresponding colony from which the cells at that position originated; and characterising a respective response of each deposited sample, in-situ on the substrate, to at least one external factor to which that sample is exposed.

Aspects of the invention extend to computer program products such as computer readable storage media having instructions stored thereon which are operable to program a programmable processor to carry out a method as described in the aspects and possibilities set out above or recited in the claims and/or to program a suitably adapted computer to provide the apparatus recited in any of the claims.

Each feature disclosed in this specification (which term includes the claims) and/or shown in the drawings may be incorporated in the invention independently (or in combination with) any other disclosed and/or illustrated features. In particular but without limitation the features of any of the claims dependent from a particular independent claim may be introduced into that independent claim in any combination or individually.

Embodiments of the invention will now be described by way of example only with reference to the attached figures in which:

Figure 1 is a simplified block schematic illustrating a genetic circuit I device testing apparatus;

Figure 2 is a simplified schematic representation of apparatus used as a cell selection platform in the apparatus of Figure 1;

Figure 3 is a simplified schematic representation of apparatus used as a cell characterisation platform in the apparatus of Figure 1

Figure 4 is a simplified drawing illustrating a characterisation element used in the characterisation platform of Figure 3;

Figure 5 is a simplified drawing illustrating how plural characterisation elements of Figure 4 may be used in the characterisation platform of Figure 3; and

Figure 6 is a simplified drawing illustrating optical characterisation of a characterisation element of Figure 4, using the characterisation platform of Figure 3.

Overview

Figure 1 is a simplified block schematic illustrating, generally at 100, a genetic circuit I device testing apparatus, or ‘test platform’, for characterising genetic circuit I device designs in cells by comparison against a pre-defined performance specification.

The test platform 100 takes as its input a liquid mixture 110 comprising genetically competent cells (those able to take up exogenous DNA) and DNA, post genetic transformation (a ‘transformation mix’). The transformation mix comprises both transformed cells (or ‘transformants’) and non-transformed cells at a ratio dependent on the efficiency of the transformation process (along with unused DNA and the like). The transformation mix will, deliberately, be formed using multiple different DNA constructs (e.g. representing different genetic circuit designs). The transformation efficiency of the transformation process is typically low (for the purpose of illustration an example efficiency of the order ~ 0.1% is assumed) leading to a correspondingly small ratio of transformed cells (i.e. those having a newly designed genetic circuit / device in them) to non-transformed cells (of the order 1:1000 in the assumed example).

The test platform 100 can be considered to be split into two main parts - a cell selection platform 120 and a cell characterisation platform 130.

The selection platform 120 selects for successfully transformed cells and sorts these selected transformed cells from the non-transformed cells. The selection platform 120 provides an output in the form of aqueous droplets each comprising a respective colony of cells grown from a single transformed cell (i.e. a monoclonal colony representing a single corresponding genetic design). It will be appreciated that whilst the exemplary droplets are aqueous they may comprise any suitable form of nonaqueous droplets including oil droplets. For each cell colony representing a respective genetic design, the cell characterisation platform 130 allows different groups of cells from that cell colony to be exposed to different concentrations of analyte, in a systematic and organised manner, to allow the corresponding genetic design to be characterised and compared to a predefined response characteristic quickly and efficiently.

As described in more detail later, the cell selection platform 120 comprises a dropletisation portion 122, incubation portion 124 and a screen and sort portion 126.

The dropletisation portion 122 forms the aqueous drops within an appropriate neutral media (typically oil). Beneficially, the volume of the droplets is carefully controlled to ensure that, statistically, each drop will carry the same average number of cells and that the average number of cells in each drop is no greater than the reciprocal of the transformation efficiency (i.e. 1000 cells per droplet in the above example where an efficiency of the order ~ 0.1% is assumed). Thus, each droplet will carry, on average, no more than a single transformant (although it will be appreciated that because the single transformant per drop is statistical, some drops will naturally carry more than one transformant and some will carry no transformants at all).

The incubation portion 124 selects for (e.g. by means of antibiotic selection) and grows, in situ in each droplet, a cell colony from any transformants in that droplet. Accordingly, the incubation portion 124 produces a flow of droplets that includes ‘empty’ droplets (e.g. droplets comprising no cells or droplets comprising only dead cells and/or un-transformed cells that have failed to result in a significant colony) and droplets comprising cell colonies grown from the transformants. Beneficially, because the droplet formation process is carefully controlled, based on statistics, to ensure an average of less than or equal to a single cell per droplet, many of the droplets output from the incubation portion 124 will be monoclonal.

The screen and sort portion 126 screens the droplets to sort the monoclonal droplets (i.e. those comprising a cell colony grown from a single transformant) from the other droplets that are either have no live cells in them. Screening may be carried out using any suitable method but, in this example, is based on optical density (or ‘turbidity’) measurements. The flow of monoclonal droplets is provided as an input, at 128, to the characterisation platform 130 whereas the other droplets are discarded as waste at 129.

As described in more detail later, the cell characterisation platform 130 comprises a store and grow portion 132, an arraying portion 134, a further incubation portion 136, a measurement portion 138, and a performance based selection portion 139.

In the store and grow portion 132, the monoclonal droplets are decoupled from the flow of droplets provided as the input 128 to the cell characterisation platform 130. Each monoclonal droplet is stored in a different respective well of an appropriately sized source plate and, if necessary, the cell colony in that droplet is allowed to grow further prior to extraction of cells from each well for arraying in the arraying portion 134.

In the arraying portion 134 a relatively small number of cells extracted from each well of the source plate are respectively deposited systematically in organised arrays on a number of solid substrates formed of an appropriate growth medium (e.g. agar or the like) on a suitable carrier (e.g. a glass slide). The position of the cells in each array is traceable back to a specific well and hence cell colony. At least some of the solid substrates on which the arrays are formed are each impregnated with analyte at a different (but known) respective concentration.

The cell micro-colonies forming the arrays on the substrates, are formed are incubated in the incubation portion 136 to promote cell growth. Thus, the cells at each position of a given array will replicate to form a different respective cell colony at that position, which is traceable directly back to the source plate well from which that colony ultimately originated. The cell colonies at each position of a given array will therefore react to any analyte impregnated into the substrate on which that array is formed, in dependence on the concentration of that analyte and on the nature of the genetic circuit / device present in the cells of that colony.

During incubation in the incubation portion 136 each cell colony is imaged at regular intervals, in the measurement portion 138, in order to perform an optical measurement of its respective response to the analyte impregnated in the substrate on which that cell colony is growing. In this example, the measurement is carried out using an optical measurement technique. The responses from the various substrates are then collated to form a respective analyte response profile for each colony. A particularly beneficial implementation of the measurement portion 138 makes use dual fluorescent reporters to respectively monitor the relative activities of each of two promoters within each cell of a given monoclonal micro-colony on the substrate (a ‘characterisation’ promotor that operates under the control of the genetic circuit being characterised and a reference promotor). The use of the two fluorescent reporters thereby allows internal ratiometric control to allow accurate quantitative characterisation of genetic circuit performance that minimises errors due to colony-to-colony variation, linked to processing variations such as, for example (but not limited to), differences in initial cell deposition number, growth rate, colony morphology, plasmid copy number, transcriptional and translational load and each colony’s oxygen availability.

Each analyte response profile is compared, in the performance based selection portion 139, to a pre-determined desired performance characteristic and the cell colony or colonies exhibiting the response profile(s) most closely matching the desired performance characteristic identified. The identified cell colonies are then traced back to the well of the source plate, and hence the original colony, from which they originated. Thus the best performing cell lines can be recovered directly from the source plate.

This split of the test platform 100 into separate selection and characterisation platforms 120, 130 allows each platform 120, 130 to employ a technique to process the cells that is tailored to the specific nature of the different processes that are used in selection and characterisation and to the constraints and requirements associated with those different processes. More specifically, the selection process employs micro-droplets and microfluidics techniques, as it is a high-speed, serial, non-organised process where cell tracking is not relevant. In contrast to this, the characterisation process is based on the formation of microarrays of cells on a suitable substrate, mainly due to the parallel, time-dependant nature of this process, and the need to keep track of the different cell lines in the platform.

It can be seen, therefore, that advantageously the way in which cell characterisation is done allows the exploration of large number of genetic circuits I devices in a consistent, quick and resource efficient manner. It allows the characterisation process to move away from a “human scale” operation to a miniaturised, low-cost option in which the dependency on large equipment and large amounts of consumables is minimised. The above platforms beneficially allow implementation as an automated or semi-automated system, with minimal human involvement, that in turn enables researchers to concentrate on the design of the genetic circuits I devices rather than the minutia of the experimental work.

The test platform is essentially based on the engineering approach of designing and building a system component (in this case a cell modified with a genetic circuit / device) to a pre-defined performance specification. The test platform provides for: the fast screening and selecting of monoclonal cells; the creation of high density arrays of discrete cell colonies on a suitable substrate; the introduction of analyte to these colonies, via pre-addition to substrate, in order to elicit a measurable response.

Cell Selection Platform

The cell selection platform 120 will now be described in more detail with reference to Figure 2 which is a simplified schematic representation of the apparatus used as the cell selection platform 120.

As explained above the cell selection platform 120 receives as its input a transformation mix comprising, as a statistical average, no more than 1 transformed cell per N cells (where 1/N gives the transformation efficiency).

The transformation process that produces transformed cells may use any of a number of different methods. One such method is illustrated, by way of example only, in Figure 2 (in simplified form). In this example, a DNA sequence 202 (e.g. representing a specific aspect of a genetic design) is artificially synthesised before being introduced, using ligation, into plasmids 210 (e.g. an expression vector) comprising one or more marker genes 204 (e.g. one or more selection markers and/or reporter markers) to form a modified plasmid 212 comprising a modified DNA sequence 206. Plasmids 212 comprising the modified DNA sequence 206 are then introduced to a liquid mixture comprising recipient cells 208. It will be appreciated that multiple different DNA constructs (e.g. representing genetic design variations) may be produced in this manner for introduction to the mixture comprising recipient cells. For example, DNA sequence variants may be introduced either through degenerate sequence introduction or alternate mutagenesis techniques, in combination with multi-fragment isothermal DNA assembly methods. The environment into which the plasmids are introduced may be designed to promote competence of the recipient cells 208 to receive the plasmid. Some of the recipient cells of the second type 208 to take up a plasmid 212 comprising the modified DNA sequence 206 through their cell walls to form a transformed cell 208'.

Typically transformation efficiency is very low (typically -0.1%) meaning that only a small fraction of the cells capable of transformation are actually transformed. A number of different approaches may be used to allow successfully transformed cells to be selected for. In this example, a selectable marker, in the form of a gene for conveying a known selectable characteristic on a cell (such as, for example, resistance to a specific antibiotic), forms part of the modified DNA sequence of the plasmid 212. Thus, successfully transformed cells 208' also acquire this selectable characteristic.

The transformed cells 208 are then isolated, in the dropletisation portion 122, by encapsulating them within aqueous droplets 214 within oil. In this example dropletisation is achieved by inputting the mix 110 of transformed and non-transformed cells into a dropletising microfluidic chip. The volume of droplets produced using this droplet formation process is controlled to encapsulate an average of roughly N cells (or fewer) within each droplet (where 1/N corresponds to the transformation efficiency). Thus, for an exemplary 0.1% efficiency, there are roughly 1000 cells per droplet of which, on average, there will be less than one successfully transformed cell. The dropletisation process involves dilution of the transformation mix to form the aqueous medium from which the drops are formed. Culture media and selection antibiotic 222 are pre-added to the aqueous solution as part of the dilution process.

The flow of droplets from the dropletisation portion 122 then passes to the incubation portion 124 where antibiotic selection 224 kills, or at least inhibits growth of, cells that have not been transformed and hence have not taken up the antibiotic resistance marker. This antibiotic selection 224 thus ensures that only successfully transformed cells grow to give rise to associated cell colonies. Following incubation, therefore, each droplet in the flow of droplets will, on average, encapsulate a colony of cells grown from a single transformed cell (where each single transformed cell may represent a different respective genetic circuit design variant). It can be seen therefore, that the statistical approach to droplet formation, beneficially promotes monoclonality within the cells in the droplets.

Statistically, some of the droplets will not include any transformed cells when formed and accordingly, post-incubation, will give rise to an ‘empty’ droplet that does not contain any live cells of significance (i.e. cells of micro-colonies grown from transformed cells) but instead only contains dead cell matter or un-transformed cells the growth of which has been inhibited via selection. Similarly, some of the droplets will be 'include more than one transformed cell when formed and accordingly, postincubation, will give rise to a ‘polyclonal’ droplet comprising a respective colony of cells grown from each transformed cell. The empty droplets are screened by the screen and sort portion 126, based on optical density measurements. Specifically, the optical density (as represented by the ‘cloudiness’ or ‘turbidity’ of the droplets) will depend on the density of cells in the droplet. Low density measurements (i.e. substantially clear droplets) are indicative of empty droplets. High density measurements (i.e. cloudy droplets) are indicative of a cell colony being present in the droplet. Hence, the droplet flow may be sorted, using conventional electromicrofluidic techniques, into an output droplet flow 128 comprising, predominantly, monoclonal cell colonies and a waste flow 129 of empty droplets. It will be appreciated that, whilst there is a finite risk that a given droplet is a polyclonal droplet, by controlling the volume of the droplet, and hence the average number of transformed cells per droplet appropriately, this risk can be reduced to an acceptable risk in the context of a given application.

Cell Characterisation Platform

The cell characterisation platform 130 will now be described in more detail with reference to Figure 3 which is a simplified schematic representation of the apparatus used as the cell selection platform 130.

As explained above the cell characterisation platform 130 receives, as its input, the output flow 128 of predominantly monoclonal droplets as screened and sorted by the cell selection platform 120. Each droplet is first decoupled from the screened flow 128, in the store and grow portion 132 of the characterisation platform 130, before being dispensed into a respective individual well of an appropriate ‘source plate’ which is a durable component providing an array of many such wells. By dispensing at the rate of one droplet in one well, each well effectively becomes the source of a different respective cell line. Each cell line is thus contained within its own well where it is allowed to grow before extraction of cells from each cell line and the formation of arrays by the arraying portion 134.

In the arraying portion 134, samples of cells from each cell line are arranged into organised arrays, via pin-spotting of the cells from the corresponding source plate well at appropriate respective locations on each of a plurality of characterisation elements 300. Efficient pin spotting may be achieved by use of an appropriate spotting pin matrix. The manner in which the arrays may be formed will now be described, by way of example only, with reference to Figures 4 and 5.

Figure 4 is a simplified drawing illustrating one such characterisation element 300 on which an organised array of samples from different cell lines has been formed. As seen in Figure 4, each characterisation element 300 comprises a carrier 410 (in this example a glass slide although any suitable carrier may be used) on which a solid substrate 420 has been formed for receiving deposited spots of cell samples during formation of the organised array. The solid substrate 420 comprises a suitable media (e.g. agar or the like) and nutrients required for keeping the cells deposited on it alive and growing. The solid substrate 420 is also typically impregnated with a required concentration of analyte. In practice, a number of such characterisation elements 300 will be formed each with a different respective concentration of analyte (or, if required, no analyte for control purposes). The deposition of the substrate is carefully controlled, and uses appropriate tooling, to ensure consistent physical characteristics across each carrier, and from carrier-to-carrier. In particular, the physical characteristics are controlled to produce homogenous substrates having a consistent thickness, a high degree of surface flatness and an even analyte distribution.

In Figure 4, microarray groups of each of a plurality of different cell lines have been formed, via pin-spotting, on the substrate 420. Whilst any suitable organised arrangement of samples from each cell line may be used in dependence on requirements, in the simplified example of Figure 4 the cell lines are organised into an array comprising two sub-arrays 425, 425' in which each sub-array comprises eight columns and five rows. Each column respectively comprises spots (in this example five) formed of cell samples from a different cell line. Thus, four spots 430, 430' in each column comprise replicates of the cell line represented in the first spot in each column. Accordingly, the example substrate 420 shown in Figure 4 carries an organised array comprising micro-array groups corresponding to sixteen cell lines with five samples of each cell line in each micro-array group.

The cells deposited on the substrate 420 of the characterising element 300 will thus mix with any analyte that has been pre-added to that substrate as they are deposited on it thereby exposing the cells to the analyte concentration on that substrate 420. Each one of the spots of cells deposited on the substrate 420 will thus lead to the formation of a different cell colony (referred to as a ‘micro-colony’ of the original cell line).

Figure 5 is a simplified drawing illustrating how plural characterisation elements 300, such as that described with reference to Figure 4, may be used to expose replicates from each cell line, to different concentrations of analyte, in an organised and traceable manner, for the purposes of efficient characterisation of the response of that cell line to the analyte for a range of different analyte concentrations.

In Figure 5, samples from each of a plurality of wells 510 of a source plate 520 are arranged, in the manner described with reference to Figure 4, on the respective substrates of each of a plurality of characterisation elements 300-1 to 300-10. Microarray groups from a given well 510 are located at the same relative position in each array on each substrate as illustrated by the various groups of arrows 530. Each substrate of a respective characterisation element 300-1 to 300-10 is impregnated with a different concentration of analyte (from 10% to 100% of a maximum concentration in the illustrated example although a range starting from 0% may also be used) and thus, samples from each cell line are exposed to a full range of analyte concentrations.

It can be seen, therefore, that the use of an organised array format such as this enables the tracking of the various micro-colonies formed from the different cell lines, and their replicates, within a given characterising element 300 and the tracing of each micro-colony back to the original cell line in the well of the source plate from which that micro-colony originated.

This approach of arraying and analyte exposure is a particularly beneficial aspect of the characterisation platform because it allows a significant reduction in the space required per individual experiment when compared with traditional techniques (e.g. based on microtiter plates or the like). The approach also simplifies greatly the process of exposing the cells to the analyte. Furthermore, as the cell lines are grouped in slides where all the micro-colonies are exposed to the same analyte concentration, it is possible to more quickly identify the micro-colonies and hence cell lines that are reacting to the analyte. In addition, the use of arrays in this manner allows efficient and reliable tracking of the different micro-colonies (of which there may be many hundreds) back to their original cell lines.

Referring back to Figure 3, the respective cells deposited at each location on the substrates of the characterising elements 300 are incubated in the incubation portion 136 and allowed to grow and react to the analyte to produce an associated response as illustrated at 310. During incubation the cell micro-colonies are imaged at regular intervals, in the measurement portion 138, in order to measure their response to the analyte.

The responses of each of the micro-colonies to the different analyte concentrations is measured using a micro array reader comprising an appropriate optical detector (typically to provide a fluorescence or light intensity measurement). Respective response profiles 320 for each of the micro-colonies can thus be constructed based on the data accrued from these measurements.

The response profile 320 for each micro-colony provides an indication of the performance of the respective genetic circuit I device integrated into the single transformed cell from which that micro-colony originated. Thus, the response profiles 320 can then be compared to a pre-defined response criterion 330 in the performance based selection portion 139 to identify the cell line(s), and hence genetic design(s), exhibiting a performance that best matches the desired response.

Cell lines identified as being successful (according to the pre-defined response criteria 330) can then be recovered directly, at 142, from the source plate, thereby providing researchers with actual cells comprising the best performing genetic designs for further research or processing.

Figure 6 is a simplified drawing illustrating operation of an optical imaging system 600 during measurement of the response of different cell lines to the analyte. As seen in Figure 6 the optical imaging system 600 comprises an imaging head 610 that is used to image a group 612 of locations each location comprising a respective monoclonal micro-colony grown from respective deposited spots of cells from the source plate. The imaging system 600 produces an image 620 of a number of such locations 630. Whilst figure 6 shows only a sub-section of the array being imaged, for illustrative purposes, it will be appreciated that the whole array may be imaged at the same time. Some of the locations 630-1 will contain a cell micro-colony that is unresponsive to the analyte in the substrate whilst other locations 630-2 will contain cell micro-colonies that respond to the cell analyte to differing degrees.

In summary, therefore, the characterisation process allows successfully transformed cells to be arranged into groups or cell lines and exposed to the different concentrations of the analyte in order to build the response profiles based on the measured response of each cell line to the different analyte concentrations. The cells are taken from the droplets, arranged in a source plate, from which cells may be taken to create arrays of cells in a substrate that exposes the array to a known analyte concentration and thus allows measurement of the response of these cells to the analyte.

Ratiometric Measurement

Optical based measurement and characterisation of responses may be carried out based on the response a particular reporter marker (such as fluorescent protein reporter or the like), that forms part of the modified DNA of transformed cells, and is expressed under the control of the genetic circuit or device that is being characterised. Whilst such characterisation may, theoretically, be carried out based on the absolute response of a genetic circuit controlled marker alone (e.g. where micro-colony to micro-colony variation across the organised arrays is minimised by tight process control) in a, particularly beneficial, example of the measurement portion 138 a ratiometric approach is used to improve characterisation accuracy. The proposed ratiometric approach has the potential to minimise errors due to a number of factors including, for example, colony-to-colony variation that may be linked to differences in initial cell deposition number, growth rate, colony morphology, plasmid copy number, transcriptional and translational load and each colony’s oxygen availability.

The ratiometric approach relies on the presence of two spectrally-distinct fluorescent protein reporters in the transformed cells. A first of these reporters is the marker that is expressed under the control of the specific genetic circuit or device that requires characterisation. The second is a ‘reference’ reporter marker that is expressed under the control of a constitutive reference promotor within the cell that typically forms part of the original recipient cells prior to transformation. By analysing the spectral makeup of a response from a particular micro-colony (i.e. at a given position in one of the organised arrays) it is therefore possible to discriminate between the responses of the different protein reporters. Thus, an internal reference fluorescence value associated with the response of the reference reporter can be measured along with a ‘characterisation’ fluorescence value associated with the absolute response of the genetic circuit controlled reporter. The respective fluorescence measurement for each of protein reporters in a given colony is substantially proportional to the number of cells in that colony. Hence, the ratio of the characterisation fluorescence value to the reference fluorescence value for a given colony will provide a normalised value that is indicative of the ‘per cell’ genetic circuit response for that colony.

It can be seen, therefore, that this internal ratiometric reference standard can be employed to allow accurate quantitative characterisation of genetic circuit performance that minimises the common-mode noise factors associated, for example, with process difference induced colony to colony variation between different sites in each array and between different characterisation elements.

To support this ratiometric technique, the different reporters selected preferably (but do not necessarily have to) meet the following criteria: (i) they provide a spectrally-distinct output to allow for independent measurement without cross-channel interference occurring; (ii) they exhibit similar biophysical properties, including approximately equivalent chromophore maturation and degradation rates, and mRNA degradation rates (although it will be appreciated that two reporters with differing degradation rates may be used, in particular if they both make protein at the same speed due to transcript numbers or ribosome binding site (RBS) strength); and (iii) they can be shown to produce equivalent results when used individually to characterise promoter activity relative to a consistent reference promoter standard.

Criteria (iii) is particularly advantageous because, for the ratiometric approach to work particularly well, the selected pair of fluorescent proteins need to give correlated proportionate responses when controlled by a range of constitutive promoters.

To aid selection of appropriate reporters, based on criteria (iii), a number of potential fluorescent protein reporters may each be combined with a reference set of constitutive promoters, spanning low to high expression levels. Each set of promoter-reporter constructs can then be characterised in individual sets, batched by reporter. A pair of fluorescent proteins can then be selected that provide sufficiently correlated responses across the range of promoters (e.g. whilst still fulfilling criteria (i) and (ii)).

Summary

In summary, it can be seen that the test platform provides an end-to-end solution to the selection and characterisation of genetic circuits. The platform takes, as inputs, a mixture of transformed and non-transformed cells, the analyte against which cells will be tested, and the response that is desired from the cell. The platform produces as outputs a response profile for each of the cell lines to have been tested and identification of cell lines having a response profile that best match the desired response.

The test platform beneficially enables researchers to explore and test a large number of genetic circuit designs in cells against a pre-defined performance specification. It achieves this, in part, by enabling automatic selection of cells that have been modified to contain the new genetic circuits (i.e. transformed cells), and the characterisation of the performance of these cells against a desired response in a consistent and rapid fashion. Researchers can then use this information to evaluate the effectiveness of the genetic designs and identify the ones that make the cells behave in the way they intended.

Particularly beneficial features of the platform include: the statistical approach to the encapsulation of cells into microdroplets which reduces the number of droplets that require processing during the selection process and improves the overall efficiency and throughput of the system; the ability to grow cell colonies in an organised manner in solid substrates, while simultaneously exposing them to a range of analytes in an orderly and controlled way; a response measurement technique based on a ratiometric approach that allows background noise to be accounted for when measuring the response of a cell line to an analyte, and enables the characterisation process to be performed using solid substrates, rather than a liquid.

Modifications and alternatives

Detailed embodiments have been described above. As those skilled in the art will appreciate, a number of modifications and alternatives can be made to the above embodiments whilst still benefiting from the inventions embodied therein.

For example, it will be appreciated that, whilst the characterisation elements are described as comprising an agar substrate on a glass slide, any suitable substrate media may be used that is suitable for keeping cells deposited on it alive and growing and any suitable carrier may be used.

Whilst the characterisation elements are described as comprising a substrate impregnated with a known concentration of a given analyte, to allow testing of the response of genetic circuits to that analyte, a given substrate may be impregnated with a plurality of different analytes. The pre-addition of two or more analytes, in predetermined concentrations, has the potential to allow more complex genetic circuits to be characterised. Similarly, different characterisation elements may each comprise a different pre-added analyte or combination of analytes. Moreover, whilst the pre-addition of an analyte into the substrate is particularly beneficial, it will be appreciated that the analyte(s) may be added later (including after the cells have been deposited).

It will also be understood that whilst the above techniques are focussed on characterising a response to the presence of one or more analytes, the characterisation may, additionally or alternatively, be based on a response to any suitable external factor (or ‘condition’) or combination of such factors including the presence of incident electromagnetic radiation (e.g. a response to a range of intensities of a predetermined wavelength of light). Moreover, whilst the invention has been described with specific reference to optical based measurements, it will be understood that the characterisation may be based on any electromagnetic radiation (including ultra-violet and/or infrared) that a reporter marker may emit.

The recipient cells into which the genetic circuits I devices are introduced may comprise any suitable cells that exhibit sufficiently rapid growth. Such cells may, for example, comprise modified bacteria cells (such as E. coli or the like) or yeast cells.

Whilst the platform is described as using antibiotic resistance based discrimination, using between transformed cells and non-transformed cells, any suitable selection marker may be used that is capable of conveying a selective advantage on the host organism post transformation. For example, an auxotrophic selectable marker in the form of an enzyme that is necessary for a particular type of cell to synthesise a particular organic compound required for its growth may be used. In the case of a yeast cell, for example, the URA3 gene may be used as a selective marker.

Where antibiotic resistance based selectable markers are used they may comprise genes encoding resistance to any suitable antibiotic (or group of antibiotics) for example one or more of the antibiotics ampicillin, chloroamphenicol, tetracycline or kanamycin.

It will be appreciated, that whilst a particular array pattern is described with reference to Figures 4 and 5 for the purposes of illustration, there are many different possible arraying patterns (that may have many more columns and/or rows and/or in which the arrangement of columns and rows is reversed with samples from the same cell line being represented in the same row and samples from different cell lines being represented in different rows) that can be employed to organise the different cell lines on the substrates of the characterisation elements. The nature of the arrays that can be used in a given scenario will, at least partly, depend on the configuration of the pin matrix used for spotting, the pitch of the different pins and the source plate, and the size of the substrate of the characterisation element.

In the selection platform, the dropletisation portion is described as producing droplets comprising, on average, a single transformed cell per droplet. It will be appreciated, however, that the dropletisation portion may be configured to produce an average of significantly less than a single cell per droplet, for example, to produce, statistically, a single cell every Z drops where Z may be greater than or equal to 2 (e.g. 3, 5, 10 ... etc.). Whilst this means that a relatively high number of drops are ‘empty’, reducing the average will have the effect of reducing the occurrence of drops having two or more transformed cells per drop, albeit at the expense of a reduced throughput of droplets comprising transformed cell.

During cell growth (before a given droplet becomes saturated with cells), polyclonal droplets, which start with more than one transformed cell, will exhibit a higher density than a monoclonal droplet (that has been growing for the same length of time). This could, in theory, represent a distinguishing feature that could be screened for to reduce the occurrence of polyclonal droplets. However, in practice, for most applications, the occasional presence of a polyclonal droplet is an acceptable cost given the other benefits offered by the process.

The measurement portion may, for example, comprise any suitable imaging device (such as a digital camera) with an associated imaging head and associated optics for capturing images. The performance based selection portion (and/or measurement portion) may comprise a computer arranged to analyse images from such imaging apparatus and to compare results of such analysis to the predetermined response characteristic.

The store and grow portion may comprise any suitable biological storage element such as a well plate, or the like. The samples may be taken using any appropriate tool such as a pipetting tool, or the like, that may be configured for taking multiple samples, substantially simultaneously from different locations (e.g. wells) of a biological storage element (e.g. well plate).

Various other modifications will be apparent to those skilled in the art and will not be described in further detail here.

Claims (33)

Claims

1. Apparatus, for a genetic material testing system, the apparatus comprising: means for receiving a mixture comprising un-transformed biological cells and transformed biological cells, wherein each transformed cell comprises at least one genetic design to be tested; and means for forming, from the mixture, a sequence of droplets, wherein the droplet formation is controlled to form droplets that comprise, on average, no more than N biological cells per droplet wherein the ratio of un-transformed biological cells to transformed biological cells in the droplet is approximately N:1.

2. Apparatus as claimed in claim 1 further comprising means for growing, in-situ in each droplet, a cell colony from any transformed biological cell in that droplet.

3. Apparatus as claimed in claim 2 wherein each transformed cell comprises at least one selection marker and wherein said means for growing is operable to select, based on the at least one selection marker and in-situ in each droplet, for any transformed biological cell in a particular droplet (and against un-transformed cells in that particular droplet).

4. Apparatus as claimed in claim 1 or 2 further comprising means for screening the sequence of droplets to sort droplets that do not comprise any transformed biological cells from droplets that do comprise at least one transformed biological cell or a colony grown from at least one transformed biological cell.

5. Apparatus as claimed in claim 4 wherein the means for screening the sequence of droplets is further configured to sort droplets that comprise a monoclonal colony grown from a single transformed biological cell from droplets that comprise a polyclonal colony grown from a plurality of different transformed biological cells.

6. Apparatus as claimed in claim 4 or 5 wherein the means for screening the sequence of droplets is operable to provide, to characterisation apparatus, a sorted sequence of droplets, wherein each of at least a majority of droplets in the sorted sequence comprises a single respective monoclonal colony grown from a single transformed biological cell.

7. Characterisation apparatus, for a genetic material testing system, the apparatus comprising: means for receiving a sequence of droplets, wherein each of at least a majority of droplets in the sequence comprises a single respective monoclonal colony grown from a single transformed biological cell comprising at least one genetic design to be tested; means for depositing at least one respective sample of cells from each colony onto a substrate comprising a material that is capable of supporting cell growth, in an organised manner, to form a predetermined pattern of samples wherein each position of the predetermined pattern is traceable back to a corresponding colony from which the cells at that position originated; and means for characterising a respective response of each deposited sample, in-situ on the substrate, to at least one external factor to which that sample is exposed.

8. Characterisation apparatus as claimed in claim 7 further comprising means for storing at least a sample of cells from each said colony in a liquid growth medium at a different respective known location, wherein each sample of cells from each colony is respectively taken from a corresponding known location for said depositing onto the substrate, and wherein each position of the predetermined pattern is traceable back to a corresponding known location of said means for storing.

9. Characterisation apparatus as claimed in claim 7 or 8 wherein the means for depositing is arranged to deposit samples from the same colony on each of a plurality of substrates for exposing the samples to a different respective external factor.

10. Characterisation apparatus as claimed in claim 7 or 8 wherein the response to at least one external factor comprises a response to at least one analyte and wherein the analyte is integrated into the substrate, prior to deposition of the deposited samples.

11. Characterisation apparatus as claimed in claim 10 wherein the means for depositing is arranged to deposit respective samples from the same colony on each of a plurality of substrates and wherein each of the plurality of substrates comprises a different respective known concentration of the at least one analyte integrated into that substrate prior to deposition of the deposited samples.

12. Characterisation apparatus as claimed in any of claims 7 to 11 wherein the pattern comprises an array having a plurality of columns and a plurality of rows.

13. Characterisation apparatus as claimed in claim 12 wherein a plurality of samples from the same colony are deposited at a plurality of different locations in the same column (or row) and a plurality of samples from different colonies are deposited at a plurality of different locations in the same row (or column).

14. Characterisation apparatus as claimed in any of claims 7 to 13 wherein each transformed biological cell from which a colony is grown comprises at least one reporting marker; and wherein the respective response of a deposited sample, to the presence of the external factor, arises from the presence of the at least one reporting marker in the transformed biological cell from which that deposited sample originated.

15. Characterisation apparatus as claimed in claim 14 wherein each transformed biological cell from which one of the colonies is grown comprises at least a first reporting marker and a second reporting marker; and wherein the characterisation means is configured to perform ratiometric characterisation of a deposited sample based on a ratio of a first response of that deposited sample arising from the first reporting marker to a second response of that deposited sample arising from the second reporting marker.

16. Characterisation apparatus as claimed in any of claims 14 to 15 wherein the, or each, reporting marker is configured to provide an electromagnetic response (e.g. a fluorescent response) and the characterisation means is configured to measure the optical response.

17. A genetic material testing system comprising, apparatus according to any of claims 1 to 6 and characterisation apparatus according to any of claims 7 to 16.

18. A method performed in a genetic material testing system, the method comprising: receiving a mixture comprising un-transformed biological cells and transformed biological cells, wherein each transformed cell comprises at least one genetic design to be tested; and forming, from the mixture, a sequence of droplets, wherein the droplet formation is controlled to form droplets that comprise, on average, no more than N biological cells per droplet wherein the ratio of un-transformed biological cells to transformed biological cells in the droplet is approximately N:1.

19. A method according to claim 18 further comprising growing, in-situ in each droplet, a cell colony from any transformed biological cell in that droplet.

20. A method according to claim 19 wherein each transformed cell comprises at least one selection marker and wherein said growing comprises selecting, based on the at least one selection marker and in-situ in each droplet, for any transformed biological cell in a particular droplet (and against un-transformed cells in that particular droplet).

21. A method according to claim 18 or 19 further comprising screening the sequence of droplets to sort droplets that do not comprise any transformed biological cells from droplets that do comprise at least one transformed biological cell or a colony grown from at least one transformed biological cell.

22. A method according to claim 21 wherein the screening of the sequence of droplets sorts droplets that comprise a monoclonal colony grown from a single transformed biological cell from droplets that comprise a polyclonal colony grown from a plurality of different transformed biological cells.

23. A method according to claim 21 or 22 wherein further comprising providing, to characterisation apparatus, a sorted sequence of droplets, wherein each of at least a majority of droplets in the sorted sequence comprises a single respective monoclonal colony grown from a single transformed biological cell.

24. A method performed by a genetic material testing system the method comprising: receiving a sequence of droplets, wherein each of at least a majority of droplets in the sequence comprises a single respective monoclonal colony grown from a single transformed biological cell comprising at least one genetic design to be tested; depositing at least one respective sample of cells from each colony onto a substrate comprising a material that is capable of supporting cell growth, in an organised manner, to form a predetermined pattern of samples wherein each position of the predetermined pattern is traceable back to a corresponding colony from which the cells at that position originated; and characterising a respective response of each deposited sample, in-situ on the substrate, to at least one external factor to which that sample is exposed.

25. A method as claimed in claim 24 further comprising storing at least a sample of cells from each said colony in a liquid growth medium at a different respective known location, wherein each sample of cells from each colony is respectively taken from a corresponding known location for said depositing onto the substrate, and wherein each position of the predetermined pattern is traceable back to a corresponding known location of said means for storing.

26. A method as claimed in claim 24 or 25 wherein the depositing comprises depositing samples from the same colony on each of a plurality of substrates for exposing the samples to a different respective external factor.

27. A method as claimed in claim 24 or 25 wherein the response to at least one external factor comprises a response to at least one analyte and wherein the analyte is integrated into the substrate, prior to deposition of the deposited samples.

28. A method as claimed in claim 26 wherein the depositing comprises deposing respective samples from the same colony on each of a plurality of substrates and wherein each of the plurality of substrates comprises a different respective known concentration of the at least one analyte integrated into that substrate prior to deposition of the deposited samples.

29. A method as claimed in any of claims 24 to 28 wherein the pattern comprises an array having a plurality of columns and a plurality of rows.

30. A method as claimed in claim 29 wherein a plurality of samples from the same colony are deposited at a plurality of different locations in the same column (or row) and a plurality of samples from different colonies are deposited at a plurality of different locations in the same row (or column).

31. A method as claimed in any of claims 24 to 30 wherein each transformed biological cell from which a colony is grown comprises at least one reporting marker; and wherein the respective response of a deposited sample, to the presence of the external factor, arises from the presence of the at least one reporting marker in the transformed biological cell from which that deposited sample originated.

32. A method as claimed in claim 31 wherein each transformed biological cell from which one of the colonies is grown comprises at least a first reporting marker and a second reporting marker; and wherein the characterisation means is configured to perform ratiometric characterisation of a deposited sample based on a ratio of a first response of that deposited sample arising from the first reporting marker to a second response of that deposited sample arising from the second reporting marker.

33. A method as claimed in claim any of claims 31 to 32 wherein the, or each, reporting marker is configured to provide an electromagnetic response (e.g. a fluorescent response) and the characterisation means is configured to measure the optical response.