METHOD AND SYSTEM FOR COLONY PICKING

The present disclosure relates to a method of colony picking. The method includes the steps of mixing a bacterial suspension with an oil-based carrier liquid for generating a plurality of droplets comprising bacteria contained in the bacterial suspension and incubating the plurality of droplets for a predetermined period of time to allow growth of bacteria within the plurality of droplets. The method further includes the steps of screening each of the plurality of droplets that flows through one or more microfluidic channels of a microfluidic device to determine an opacity degree of each of the plurality of droplets, wherein the opacity degree is indicative of colony formation in the plurality of droplets, and sorting the plurality of droplets based on the opacity degree of each of the plurality of droplets.

FIELD OF INVENTION

The present invention relates broadly, but not exclusively, to a method and system for colony picking.

BACKGROUND

The process of introducing DNA (and other similar polynucleotides) into host cells is a key aspect of recombinant DNA technology. The process by which polynucleotides are introduced into host cells is called transformation. Bacterial cells generally remain the preferred hosts for the majority of recombinant DNA experiments and genetic engineering manipulations.

Conventional methods of culturing and selecting bacterial cells typically involve culturing transformed bacterial cells on antibiotic-containing agar plates. Only bacterial cells that contain the introduced DNA would be able to divide and grow and form colonies in the presence of the antibiotic. Colony picking and/or colony counting will then be performed before carrying out downstream procedures.

Colony picking and counting procedures are tedious and time consuming. Efforts have been made to automate these procedures mainly through the development of automated colony pickers. By using image analysis techniques and internal algorithms to differentiate actual colonies from background noise caused by bubbles and colony irregularities, the colonies formed on the agar plates can be counted and picked by robotic-controlled sterilized pins and segregated into individual wells (typically of 96 and 384 well plates) for culturing. However, the use of automated colony pickers is limited by the high costs of the equipment, the amount of reagents used (which is similar to the amount used in manual methods), the risk of contamination, and the level of equipment maintenance required. In addition, the low throughput level (on average about 1000 colonies/hour) makes it difficult for the automated colony pickers to keep up with the ever expanding need for large scale genetic constructions.

Thus, there is a need for an automated method of colony picking and/or counting that circumvents the problems associated with the existing colony picking and/or counting procedures.

A need therefore exists to provide a system and method that seek to address at least one of the problems above or to provide a useful alternative.

SUMMARY

According to a first aspect of the present invention, there is provided a method of colony picking, the method comprising the steps of:mixing a bacterial suspension with an oil-based carrier liquid for generating a plurality of droplets comprising bacteria contained in the bacterial suspension;incubating the plurality of droplets for a predetermined period of time to allow growth of bacteria within the plurality of droplets;screening each of the plurality of droplets that flows through one or more microfluidic channels of microfluidic device to determine an opacity degree of each of the plurality of droplets, wherein the opacity degree is indicative of colony formation in the plurality of droplets; andsorting the plurality of droplets based on the opacity degree of each of the plurality of droplets.

According to a second aspect of the present invention, there is provided a system for colony picking comprising:a droplet generator for mixing a bacterial suspension with an oil-based carrier liquid to generate a plurality of droplets comprising bacteria contained in the bacterial suspension;an incubator for culturing the plurality of droplets for a predetermined period of time to allow growth of bacteria within the plurality of droplets;a microfluidic device comprising one or more microfluidic channels for receiving the incubated droplets, wherein each of the plurality of droplets flows through the one or more microfluidic channels consecutively;an optical device for screening each of the plurality of droplets that flows through the one or more microfluidic channels to determine an opacity degree of each of the plurality of droplets, wherein the opacity degree is indicative of colony formation in the plurality of droplets; anda sorting module configured to sort the plurality of droplets based on the opacity degree of each of the plurality of droplets.

DETAILED DESCRIPTION

FIG.1Ashows a diagram100illustrating the process of preparing droplets102containing bacteria in accordance with an example embodiment. A bacterial concentration including a culture medium is prepared and incubated for a predetermined period. The bacterial concentration is then diluted with a culture medium that contains a component suitable to exert a selection pressure on genetically modified bacteria (e.g. antibiotic) to form a bacterial suspension at a predetermined concentration.

Next, each bacterium in the bacterial suspension is encapsulated in single-cell droplets and incubated for a predetermined period before being screened by a microfluidic system for droplets that include microbial colonies. Details of the system and method for colony picking in accordance with an example embodiment are explained in further detail below.

A bacterial suspension104is prepared in a first container by diluting a bacterial concentration with a culture medium to a predetermined concentration (e.g. 100 bacteria/ml). The culture medium contains an antibiotic to exert a selection pressure on the bacteria. For example, the initial bacterial concentration containsEscherichia coliDH10β (New England Biolabs) cells that has undergone transformation and grown in Lysogeny broth (LB) medium without antibiotics in the recovery phase. The bacterial concentration is then diluted with the LB medium with antibiotic kanamycin (50 ug/ul) (Sigma) into a bacterial suspension at a predetermined concentration.

The radius of the droplets and the concentration of the bacterial suspension are selected to achieve 1 bacterium per droplet for single-cell encapsulation. Specifically, to achieve an average of 1 bacterium per droplet, the concentration of the bacterial suspension is determined based on the desired radius of the droplets according to the formula

where r is the radius of the droplets in μm.

As shown in the enlarged view of the droplet generator114inFIG.1A, the bacterial suspension104and the oil-based carrier liquid106flows in a direction (shown with arrows116inFIG.1A) towards to a junction, and are mixed to form a plurality of droplets102with the bacterial suspension104being encapsulated by the oil-based carrier liquid106. Due to the selection of the radius of the droplets and concentration of the bacterial suspension104, the generated droplets102are mostly empty or contain single bacterium. The generated droplets102then flow to an outlet118in the droplet generator114and collected in a third container.

Next, the collected droplets102are placed in an incubator for a predetermined temperature and a predetermined period of time (e.g. 22° C. for 24 hours). Due to the presence of antibiotic in the culture medium of the bacterial suspension104, only the successfully transformed bacteria which have gene that is resistant to the antibiotic would proliferate and form a monocolony in the droplets102during the incubation process120.

FIG.1Bshows a diagram122illustrating the process of screening the droplets102ofFIG.1Afor a presence of colony. Upon completion of the incubation process, a pump124is used to administer the incubated droplets102to a microfluidic device126for screening and sorting of individual droplets102. As shown in the enlarged view of the microfluidic device126inFIG.1B, the microfluidic device126includes one or more microfluidic channels128that allow each of the plurality of droplets102to flow through consecutively for the screening and sorting processes to take place. It will be appreciated that the droplets102may be collected and incubated in a reservoir (e.g. 1 cm3) that is integrated with the droplet generator114and the microfluidic device126. The reservoir can include a valve operable to allow the droplets102to be drained out of the reservoir and flow into the microfluidic channel128without using the pump124.

When the droplets102pass through the microfluidic channels128, an optical device having an objective with a field of view130is used to screen each droplet102by measuring a bright field intensity associated with the droplets102to determine an opacity degree of the droplets102. The opacity degree of the droplets102is indicative of colony formation in the plurality of droplets. The optical device generates a signal based on the measured bright field intensity and transmits the signal to a data acquisition unit for comparison with a predetermined threshold by a sorting module132.

For example, the microfluidic device126is used for screeningEscherichia colicell that harbor an antibiotic resistance plasmid. For cells that have successfully harbored the transformed plasmid, they would grow in the antibiotic-laden medium and expand into a bacterial colony inside the droplets102, thereby increasing the opacity of the droplets102and causing low bright field signal to be generated by the optical device. Opaque droplets102awhich contain the cells could be subsequently sorted away from empty droplets102bthat have low opacity.

The optical device may have two or three components: (1) a light source, (2) a detecting unit, and (3) an amplifying unit to multiply signal from the detecting unit if the signal is weak. In an example, the light source is a halogen light (Leica, Germany), the detecting unit is an inverted microscope DMi8 (Leica, Germany), and the amplifying unit is a photomultiplier tube (PMT) (Hamamatsu, Japan).

Droplets102containing bacteria are administered into a long microfluidic channel with a predetermined dimension (e.g. 80×80×90 μm3) at a predetermined flow rate (e.g. 4 μl/min) with a pump (e.g. a syringe pump, Harvard Apparatus, USA). An observation window (e.g. an area of 211.2×211.2 μm2) was continuously shined with halogen light for detecting the bright field intensity when the droplets102passed through the microfluidic channel128. The signal generated based on the bright field intensity is amplified with PMTs and logged into MATLAB for data processing.

The sorting module132compares the generated signal from the optical device with a predetermined threshold and transfers the droplets102based on the opacity degree of each droplet102. Specifically, if the intensity signal is lower than the predetermined threshold which suggests that an opaque droplet102awith successfully transformed bacteria passes through, the sorting module132is activated to pull the opaque droplets102ato a collection outlet. On the other hand, if the intensity signal is higher than the predetermined threshold, the sorting module132would allow the empty droplets102bto passively flow towards a waste outlet.

In an example, the sorting module132includes electrodes to synthesize a non-uniform electrical field to pull the opaque droplets by dielectrophoresis. Other examples to actively sort the opaque droplets102ainclude (1) operating a membrane valve to pump air/fluid to block a particular channel, (2) generating surface acoustic wave (SAW) to push the opaque droplets102aand (3) operating a dynamic fluid switch to inject pulse of fluids from side channels to push the opaque droplets102a. Alternatively, the opaque droplets102acan be passively sorted (1) based on the content and mass of droplets102using gravity or (2) based on difference in size with deterministic lateral displacement (DLD).

FIG.1Cshows a graph134illustrating an example of a generated signal in the screening process of the droplets102ofFIG.1B. Intensity (a.u.) is plotted on the y-axis and time (s) is plotted on the x-axis. In other words, the graph134shows the changes in the intensity signal captured by the objective of the optical device over time in the field of view130at the microfluidic channel128. As shown in the graph, a threshold (represented as reference numeral136inFIG.1C) is predetermined between 100 a.u. and 150 a.u. on the y-axis. The sorting module is configured to compare the intensity signal with the predetermined threshold136and transfer the droplets102to different groups based on the comparison of the intensity signal with the predetermined threshold136. In the example shown inFIG.1C, the intensity signal drops around time 0.05 s, suggesting that an opaque droplet102awith successfully transformed bacteria is passing through. In that case, the sorting module is activated to pull the opaque droplets102ato a collection outlet.

Bacterial colony picking/counting remains an essential but mundane task for microbiologists. Efforts have been made to automate the process of colony picking/counting on the plate through the development of automated colony pickers. Nevertheless, automated colony pickers are still limited by the high cost, the risk of contamination, heavy maintenance requirement, and its low throughput.

The system and method for colony picking of the example embodiments involve using integrative microfluidic modules for automation in screening colony formation in droplets102that contain bacteria and antibiotic after an incubation process based on the opacity of the droplets. The system and method may also be used to screen enzymes-mutant library for colonies containing unique protein sequences generated by site-directed mutagenesis. Advantageously, the method of the example embodiments can be used for screening for bacteria without any reporter or label since the bacterial colonies are picked based on the opacity of the droplets102.

The system and method of the example embodiments can facilitate significantly greater throughput (1000 Hz or >million droplets per day) by allowing automated colony picking process while significantly reducing cost (e.g. cost in bio-reagents), time and variability. The bridging of the gap between post-transformation and colony screening processes allows complete chemical isolation between the processes. This may minimise contamination among different colonies and reduce the space required for the colony picking process, as compared to the conventional method which uses agar plates.

FIG.2shows 2 sets of graphs (a, b, c and d) including screening results associated with droplets with diameters of 40 μm and 100 μm respectively in accordance with an example embodiment. Depending on the bacteria strains which would result in different proliferation rate, cell size, cell transparency, etc., the degree of change in the opacity of the droplets during an incubation process may vary from one bacterium to another. Thus, droplet size would need to be optimized to yield a more accurate selection.

In the graphs a, b, c and d, opacity detected by PMT (a.u.) is plotted on the y-axis and bacteria concentration (/ml) is plotted on the x-axis for an experiment that involvesEscherichia coli. As shown in graphs a and b which illustrate the screening results of 40 μm droplets, opacities (mean opacity in graph a and distribution of opacity in graph b) are compared among droplets with cell culture medium (no cell, negative control), single cell cultured for 4 hours (8.75E7 cell/ml), 6 hours (3.1E8 cell/ml), 7 hours (3.66E8 cell/ml), and stock cell suspension (1.01E10 cell/ml, positive control). There is substantial overlapping in the opacity signals of the droplets of all the different concentrations including droplet that contain only medium and droplets that contain cells in high concentration.

As shown in graphs c and d which illustrate the screening results of 100 μm droplets, opacities (mean opacity in graph c and distribution of opacity in graph d) are compared among droplets with cell culture medium (negative control), single cell culture for 24 hours, and stock cell suspension (1.01E10 cell/ml, positive control). As shown in graph d, there is negligible overlap in the opacity signals of the droplet that contain medium and droplets that contain cells. The wider the range of the overlapping in the opacity signal such as that shown in graphs a and b for 40 μm droplets would cause difficulty in setting the threshold for sorting the droplets. Thus, forEscherichia coli, a larger droplet may facilitate the screening process as large droplets allow more cells to grow and thus a more precise range in opacity which can help in more accurately differentiating droplets containing different concentrations.

A suitable threshold can be set for sorting the droplets with successfully transformed cells from others depending on the applications of the experiments. For example, in graph d, line 1 represents a lower threshold to capture all the positive droplets. Although this allows a higher number of positive droplets to be collected, the collected droplets may contain more false positive (i.e. empty droplets). Line 2 represents a higher threshold to capture droplets with as little negative droplets as possible. Although this reduces the number of negative droplets to be collected, it may compromise the number of positive droplets collected.

FIG.3shows a flow chart300illustrating a method for colony picking in accordance with an example embodiment. At step302, a bacterial suspension is mixed with an oil-based carrier liquid for generating a plurality of droplets comprising bacteria contained in the bacterial suspension. At step304, the plurality of droplets is incubated for a predetermined period of time to allow growth of bacteria within the plurality of droplets. At step306, each of the plurality of droplets that flows through one or more microfluidic channels of a microfluidic device is screened to determine an opacity degree of each of the plurality of droplets, wherein the opacity degree is indicative of colony formation in the plurality of droplets At step308, the plurality of droplets is sorted based on the opacity degree of each of the plurality of droplets.