KR20150143177A

KR20150143177A - Hydrogel-in-oil emulsion containing cell, method for in vitro compartmentalization and ultra-high throughput screening of novel enzyme

Info

Publication number: KR20150143177A
Application number: KR1020140072359A
Authority: KR
Inventors: 송재준; 최종현; 고경철; 이빛나
Original assignee: 한국생명공학연구원
Priority date: 2014-06-13
Filing date: 2014-06-13
Publication date: 2015-12-23
Also published as: WO2015190730A1; KR101663556B1

Abstract

The present invention relates to hydrogel-in-oil emulsions, extracellular compartments ( in vitro compartmentalization; IVC) method and novel enzyme ultra-high-throughput screening (uHTS). The diffusion of the reaction product between the cells and the substrate in the hydrogel is prevented and the fluorescence intensity is strong, so that it can be very usefully used for FACS analysis.
In addition, the emulsion according to the extracellular compartmentalization (IVC) method of the present invention can be stably stored for a long period of time, and the cells can be cultured, and thus the cells can be proliferated from a single cell to two or three cells.

Description

The present invention relates to a hydrogel emulsion, an extracellular compartmentalization method, and a novel enzyme high-speed screening method containing cells, and more particularly, to a method for preparing a hydrogel-in-oil emulsion containing cell, a method for in vitro compartmentalization and ultra high throughput screening of novel enzyme,

The present invention relates to hydrogel-in-oil emulsions, extracellular compartments ( in vitro compartmentalization; Hereinafter referred to as 'IVC') and ultra-high-throughput screening (hereinafter referred to as 'uHTS').

A commonly known method for discovering new enzymes is to detect enzyme activity based on an agar plate or a microtiter plate (Chem. Biol . 2004, 11: 981-990; Enz. Microbiol Technol., 2004, 34: 429-436). The number of libraries that can be screened per day does not exceed about 10 ³ to 10 ⁵ despite the development of an automated system.

However, along with the development of the industry, it is urgently required to develop a technique capable of performing ultra high-throughput screening (hereinafter referred to as "uHTS") more efficiently than the screening method that can be currently processed (Nat. Biotechnol. 2001, 19: 537-542; Curr. Opin. Biotechnol., 2004, 15: 323-329; Nat. Chem. Biol., 4: 290-294).

Recently, the possibility of fluorescence-activated cell sorting (FACS) as a promising tool for uHTS has been proposed, which can analyze 10 ⁷ cells / hour or more (Curr. Opin. Chem. Biol 2005, 9, 210-216; Chem Biol., 2005, 12: 1255-1257). FACS analysis is capable of efficiently screening large libraries and has potential as an effective tool for the analysis of directed evolution.

However, in order to screen the enzyme using FACS, which is a fluorescence detection base, it is only possible if a cell expressing a specific enzyme decomposes a substrate and the fluorescent product is adsorbed on the cell surface, Biotechnol 2000, 18: 1071-1074; PNAS 2005, 102: 6855-6860; PNAS 2005, 120: 10082-10087). However, most substrates enter the cell and react with the enzyme, resulting in a rapid disruption of the fluorescent product produced outside the cell (Appl. Biochem. Biotechnol., 161, 301-312). The biggest problem is that it is very difficult to find a substrate. In addition, when an expressed enzyme is secreted outside the cell and an enzyme reaction occurs outside the cell, it is impossible to detect a specific target enzyme.

In vitro compartmentalization (IVC) techniques using water-in-oil emulsions (hereinafter referred to as 'w / o') have been reported as a method for solving the above problems (EMBO J 2003, 22: 24-35; Appl. Microbiol. Biotechnol., 2011, 89: 1453-1462).

IVC was originally developed as a technique for generating artificial cells for directed evolution of proteins (Nat. Methods 2006, 3: 561-570), using a single gene And its transcriptional and detoxification constructs are surrounded by a specific substrate to form an artificial membrane and the fluorescence signal according to the expression of a single gene can be analyzed by FACS (Nat. Methods 2006, 3 : 561-570; Appl. Microbiol. Biotechnol., 2011, 89: 1453-1462). These IVC techniques can be applied to isolate single cells independently and provide conditions under which FACS can be analyzed by allowing fluorescent products to remain in artificial cells due to substrate degradation (Nat. Methods 2006, 3: 561-570).

However, limitations of IVC technology have been identified in the search for novel enzymes using conventional IVC techniques of water-in-oil emulsions (w / o). The limitations of IVC technology are limited by specific enzyme reactions The very small molecules of the fluorescent substance decomposed from the substrate pass through the oil film and diffuse into the outer aqueous solution layer within a short period of time.

That is, there is a problem in that the fluorescent intensity in the IVC is lowered so that the FACS detection is limited, and the formed emulsion is easily destructed, making it difficult to store for a long period of time. IVC technology which can solve low sensitivity and instability which is a problem of IVC using such conventional oil-in-oil emulsions (w / o) is desperately needed.

In order to solve the above-mentioned problems, an object of the present invention is to provide a method for producing a cell-containing oil-in-oil emulsion (hereinafter referred to as 'h / o'), an extracellular compartmentalization method, (IVC) method using a hydrogel instead of water in order to prevent the diffusion of the substance in the emulsion, and a process for producing an aqueous solution containing a single cell Hydrogel-in-oil emulsions and a novel enzyme-based ultra-rapid screening method using the emulsion.

The present invention relates to hydrogel-in-oil emulsions, extracellular compartments ( in vitro compartmentalization; IVC) method and novel enzyme ultra-high-throughput screening (uHTS).

More particularly, the present invention relates to a method for producing a hydrogel-forming material, comprising: (a) preparing a liquid medium comprising a hydrogelation-forming material; (b) adding the prepared liquid medium to cells to form a suspension; (c) adding the suspension of step (b) to an oil surfactant mixture; And (d) after step (c), homogenizing and gelating the suspension to form a hydrogel-in-oil emulsion in which the cells are contained Extracellular compartmentalization ( in vitro compartmentalization; IVC) method.

In addition, the present invention provides hydrogel-in-oil emulsions containing cells prepared by the extracellular compartmentalization (IVC) method.

In addition, the present invention provides a novel enzyme-based ultra-rapid screening method by a fluorescence-activated cell sorting (FACS) method using hydrogel-in-oil emulsions in oil containing the cells.

The present invention relates to hydrogel-in-oil emulsions, extracellular compartments ( in vitro compartmentalization; IVC) method and novel enzyme ultra-high-throughput screening (uHTS). Since the small molecule fluorescent substance produced in the intracellular enzyme and the substrate reaction using the hydrogel-in-oil emulsions containing cells according to the present invention can not pass through the hydrogel layer, It is very useful for FACS analysis because it can reduce the loss.

In addition, the emulsion containing the cells according to the extracellular compartmenting method of the present invention can be stably stored at 2 to 4 캜 for 1 to 10 days. Since the cells can survive and proliferate in the emulsion hydrogel, When the cells are proliferated and analyzed by two or three cells, there is an advantage that the degradation of the substrate can be confirmed more smoothly.

Figure 1 shows the size of the microbeads formed according to the homoginization time and was 8,000 for 30 (A), 60 (B), 90 (C), 120 (D), 150 0.0 > rpm. < / RTI >
FIG. 2 shows the size of the microbeads formed according to the homoginization time, and it was 9,500 for 30 (A), 60 (B), 90 (C), 120 (D), 150 0.0 > rpm. < / RTI >
FIG. 3 shows the size of microbeads formed according to the homoginization time, which is 13,500 (A), 60 (B), 90 (C), 120 0.0 > rpm. < / RTI >
Figure 4 shows the size of the microbeads according to the rpm of the homogenizer and the operating time.
FIG. 5 is an analysis of the size of a microbead using a particle size analyzer. FIG.
6 shows h / o emulsion formation using GFP cells in a cell culture medium with OD ₆₀₀ values of 0.2 (A), 0.4 (B), 0.6 (C), 0.8 (D) The optical and fluorescence images are superimposed, and the right is a fluorescence image.
FIG. 7 shows the formation of h / o emulsion using GFP cells, which was observed immediately after the preparation of the h / o emulsion (A) and observed after incubation for 24 hours (B). The left side shows the optical and fluorescence images superimposed, and the right side shows the fluorescence image.
Fig. 8 is a photograph of a microbead containing GFP cells, showing optical images and fluorescence images superimposed on the left, and fluorescence images on the right.
FIG. 9 shows FACS analysis results of gel beads containing GFP cells. (NF: no fluorescence, LF: low fluorescence, HF: high fluorescence) of the microbeads (A), the microbeads (B) containing the GFP cells, and the fluorescence intensity of the microbeads.
FIG. 10 is a photograph (NF: no fluorescence, LF: low fluorescence, HF: high fluorescence) after UV light observation after FACS analysis and sorting by fluorescence intensity and culturing.
Fig. 11 shows the h / o emulsion containing 1 mM MU. The left side shows the h / o emulsion and the right side shows the microbe image after 20 minutes after the oil removal of the h / o emulsion.
Figure 12 shows the results of FACS analysis of microbeads containing 1 mM MU. The above figure is a negative control, and the lower drawing is a microbead containing 1 mM MU.
13 is a photograph of microspheres containing exocellulase-expressing cells and MUG ₂ reacted for 24 hours. Wild-type E. coli XL1-blue cells (A), and exocellulase-expressing cells (B).
14 shows the result of FACS analysis after 24 hour reaction of exo-cellulase expressing cells and h / o microbeads containing MUG ₂ . Above: Wild type E. coli XL1-blue cells, and the results are as follows: Observation of microbeads containing exo-cellulase-expressing cells.
Figure 15 shows the expression of wild type E. coli XL1-blue cells and exo-cellulase-expressing cells were incubated for 24 hours with 9: 1 microbeads, and analyzed by FACS. The stomach is a negative control, and the bottom is E. coli The microbeads were prepared by mixing XL1-blue cells and cells containing exo-cellulase at a ratio of 9: 1.
FIG. 16 is a photograph showing the result of culturing the sorted fraction, showing the photograph on the left side of the UV transilluminator and the right side showing the fluorescence value measurement using Victor.
FIG. 17 shows the results of observation of hydrogel beads containing GFP cells using PEGDA. The left side shows the optical and fluorescence images superimposed, and the right side shows the fluorescence images.
FIG. 18 is a result of FACS analysis of GFP cells in hydrogel beads prepared using PEGDA, wherein the stomach is a negative control, and the following is a result of analysis of hydrogel beads containing GFP cells.
19 shows the result of observing the number of microbeads using a hemocytometer. (A) is a schematic diagram of a hemacytometer, (B) is a microbead after removal of oil film with beads of h / o emulsion, PBS, hexane and mineral oil.
20 is an optical and fluorescence photograph of a water-in-oil-in-water (w / o / w) dual emulsion, And the red arrow indicates GFPuv expressed in Escherichia coli (the scale bar is 50.0 mu m).
21 is a micrograph of an emulsion containing 4-methylumbelliferone (hereinafter, referred to as 'MU'), wherein the left is a photograph in which optical and fluorescence photographs are overlapped and the right is a fluorescence photograph (scale bar is 50.0 μm to be).
22 is a photograph of an emulsion produced by incorporating 4-methylumbelliferyl-β-D-cellobioside (hereinafter referred to as 'MUG ₂ '), (A) and emulsion were prepared and reacted for 24 hours. Microscope (B) shows a photograph on the left by an optical microscope and on the right by a fluorescence microscope (scale bar is 50.0 μm).
Figure 23 shows the diffusion of MU out of the IVC of the w / o / w emulsion, showing photographs immediately after the w / o / w emulsion containing MU and the w / o / w emulsion not containing 1: (B) observed after 20 minutes from the mixing of (A). The left side is an optical microscope photograph and the right side is a fluorescence microscope photograph (scale bar is 50.0 μm).
24 shows the result of FACS analysis of the w / o / w emulsion using MU, wherein the upper drawing shows the negative control group and the lower drawing shows the 1 mM MU positive control group.
FIG. 25 shows the result of FACS analysis of the w / o / w emulsion using GFP cells, wherein the upper drawing shows the negative control group and the lower drawing shows the GFP expression positive control group.

The method may further comprise, after the step (d), culturing the cells contained in the emulsion at 30 to 37 캜 for 1 to 24 hours, wherein in the step (b), the prepared liquid medium is added to the cultured cells In the step of adding a suspension to form a suspension, a substrate is further added to form a suspension.

The substrate is a substance that reacts with an enzyme in a cell and is characterized in that a fluorescence signal can be confirmed by an enzyme and a substrate reaction.

The hydrogelation-forming material may be selected from the group consisting of agarose, chitosan, alginate, polyethylene glycol, polyethylene glycol diacrylate (PEGDA) polyvinyl alcohol, Polyacrylamide, polyurethane, polypropylene glycol, ployvinylpyrrolidone, xanthan, hyaluronan, gelatin, polyacrylic acid (such as polyacrylic acid, more preferably 2 to 4% (w / v) low melting agarose or polyethylene glycol (w / v) selected from the group consisting of polyacrylic acid, carboxymethyl celluose and collagen, But is not limited to, polyethylene glycol diacrylate (PEGDA), which can form a hydrogel and is free from cytotoxicity If the quality can be used mubang anything halfway. The polyethylene glycol (hereinafter referred to as 'PEG') is a biocompatible material having excellent biocompatibility, rigidity and non-reactivity characteristics among pylymeric materials and can be used for cell encapsulation, microarray, and drug delivery (Biomed Mater. 2009, 4: 011001-011008; Materials 2009, 2: 577-612). Thus, PEG-based polymers are highly desirable materials for forming biocompatible hydrogels as hydrophilic polymers.

The homoginization is preferably homogenized for 30 to 180 seconds at 8,000 to 13,500 rpm using a homogenizer. However, the method for forming the microbeads is not limited thereto and any method may be used .

The emulsion is preferably a micro-bead having an average diameter of 2 to 50 탆, more preferably a micro-bead having an average diameter of 10 to 30 탆, and most preferably a single cell is suitable for IVC in a micro- Size micro-bead having a diameter of 20 mu m.

Preferably, the cell is a single cell, but is not limited thereto.

The size of the hydrogel-in-oil emulsions in the cells containing the cells has an average diameter of 2 to 50 mu m and is characterized by being able to be stored at 2 to 4 DEG C for 1 to 10 days, The culture is preferably carried out at a temperature of 30 to 37 DEG C for 1 to 24 hours, but is not limited thereto.

(A) adding a substrate to a hydrogel emulsion in an oil containing cells;

(b) reacting the substrate added in the step (a) with the enzyme expressed in the cells in the hydrogel; And

(c) confirming the reaction result of the step (b) by a fluorescence-activated cell sorting (FACS) method.

Since the hydrogel emulsion in oil has high viscosity and can not be analyzed by FACS, it is necessary to remove the oil and hydrogel-in-oil (oil-in-oil) emulsion. Preferably, the oil is removed using any one selected from the group consisting of mineral oil, hexane, and 2-propanol. More preferably, mineral oil is used, but not limited thereto. By observing the number of colonies produced by culturing the oil-removed beads with an organic solvent such as mineral oil, hexane and 2-propanol, an organic solvent more safe for cell survival can be obtained. .

Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples. It is to be understood by those skilled in the art that these examples and comparative examples are merely intended to explain the present invention more specifically and that the scope of the present invention is not limited thereto.

Example 1. IVC production of agarose beads for ultra-rapid search of novel enzymes and FACS analysis

(1) h / o Emulsion Formation and Microbead Resize

The hydrogel type emulsion using agarose was named 'h / o emulsion'. It was confirmed that the size of the gel beads was controlled by the rpm of the homogenizer and the time for homogenization.

LB liquid medium containing 3% (w / v) low melting agarose (3% (w / v) low melting agarose) was sterilized and stored in an incubator at 65 ° C. 1 ml of the stored agarose-LDB medium solution was taken and placed in a 1.5 ml microtube and placed in a hit block set at 40 ° C so that the temperature of the solution became 40 ° C. 200 占 퐇 of an agarose-LB medium solution at 40 占 폚 was sampled and placed in 400 占 퐇 of an oil surfactant mixture (2.9% ABIL EM90 in mineral oil). Homogenization was carried out using a homogenizer while being fixed to a hit block at 40 ° C.

The rpm of the homogenizer was 8,000, 9,500 and 13,500 rpm, and homogenized for 30, 60, 90 120, 150 and 180 seconds per rpm. After homogenization, the mixture was allowed to stand on ice for 5 minutes to gel.

The completed h / o emulsion was confirmed through a microscope (Figs. 1 to 3). As the homoginization rpm and time increased, the size of beads gradually decreased and the number of beads increased.

The size of the prepared microbeads was determined as an average diameter, and the result was shown in a graph (FIG. 4).

In this embodiment, the conditions of 9,500 rpm and 1 minute for forming a 20-μm diameter were selected for IVC in a bead, and a uniform average 20 μm average size was measured using a particle size analyzer (MasterSizer 2000, Malvern Instruments Ltd.) Diameter microbeads were fabricated (Fig. 5).

(2) GFP Optical density of cells ( optical density ; Below OD H / o according to emulsion formation

The ratio of microbeads containing cells in h / o emulsion formation according to OD ₆₀₀ of each of the cells was determined. The experiment was carried out in the following manner.

LB liquid medium containing 3% (w / v) low melting agarose was sterilized and stored in an incubator at 65 ° C. 1 ml of the stored agarose-LDB medium solution was taken into a 1.5 ml microtube and placed in a heat block set at 40 ° C so that the temperature of the solution became 40 ° C. By a cell sorting (GFPuv E. coil) in the centrifugal each OD _{(OD 600 = 0.2, 0.4,} 0.6, 0.8, 1.0) each of the cell culture taken to 6,000rpm 1㎖ Align to 5 minutes incubation, the upper layer The solution was carefully removed and the cells were washed with 1X PBS. The cells were resuspended in 1 ml of agarose-LB medium solution at 40 ° C., 200 μl of which was collected and placed in 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil) gave. The mixture was fixed on a heat block at 40 ° C. and homogenized at 9,500 rpm for 1 minute using a homogenizer. The mixture was allowed to stand on ice for 5 minutes for gelation. The h / o emulsion thus prepared was confirmed by optical and fluorescence microscopy (Fig. 6). When the cell culture medium having an OD value of 1 was used, it was confirmed that beads containing cells were present at a ratio of about 80%. An appropriate OD value was selected according to the purpose of the experiment.

(3) GFP Cell-based h / o emulsion Production and Bead Observation of cell proliferation inside

An h / o emulsion containing GFP (green fluorescent protein) cells was prepared by the following method. LB liquid medium containing 3% (w / v) low melting agarose was sterilized and stored in a 65 ° C incubator. 1 ml of the stored agarose-LDB medium solution was taken in a 1.5 ml microtube and placed in a hit block set at 40 ° C so that the temperature of the solution became 40 ° C. 1㎖ of cell culture broth (GFPuv E. coil cells, OD ₆₀₀ = 0.6) was collected and centrifuged for 5 minutes at 6,000rpm, and the supernatant was carefully removed and the cells were washed with 1 × PBS. The cells were resuspended in 1 ml of agarose-LB medium solution at 40 ° C., 200 μl of which was taken out and put into 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil) gave. The resulting h / o emulsion was immobilized on a hit block at 40 ° C for 1 minute at 9,500 rpm using a homogenizer, allowed to stand on ice for 5 minutes, And confirmed with a microscope. It was confirmed that h / o emulsion containing single GFP cells was successfully generated in each microbead.

After the h / o emulsion was prepared, it was incubated at 37 ° C for 24 hours to confirm survival and proliferation of the cells in the agarose gel. After the h / o emulsion was prepared and after 24 hours, it was confirmed by fluorescence microscopy to determine the proliferation. Observation revealed that the cells proliferated 24 hours later (Fig. 7). Therefore, it was concluded that the number of single cells could be increased to 2 ~ 3, and more smooth substrate decomposition could be achieved.

(4) GFP Cell-containing Microbead FACS analysis

For the FACS analysis, oil removal was performed because the oil film of the h / o emulsion had to be removed and agarose gel bead particles had to be obtained.

100 의 of h / o emulsion containing GFP cells was collected and placed in 1 ml of PBS (containing 10% hexane) and vortexed for 10 seconds. After centrifugation at 5,000 rpm for 30 seconds, the supernatant was carefully removed. 1 ml of PBS was added, the microbeads were carefully resuspended, centrifuged at 5,000 rpm for 30 seconds, and the supernatant was carefully removed. The microbeads were carefully resuspended in 1 ml of PBS and transferred to a new microtube.

After removing the oil by the above method, the microbeads containing the recovered GFP cells (FIG. 8) were analyzed by FACS and significant results were obtained.

For FACS analysis, blue laser suitable for GFPuv wavelength band (Excitation 395nm, Emission 509nm) was used. The difference in fluorescence intensity between beads containing and without GFPuv cells was clearly distinguished (FIGS. 9A and 9B). 9C). The aligned beads were spread on an LB agar plate and cultured, and the presence of beads in GFPuv cells was confirmed (FIG. 10). A group showing high fluorescence intensity as compared with the control group (GFP-free gel beads) was sorted and cultured. As a result, GFP colonies were formed, and groups having fluorescence intensities similar to those of the control group were sorted and cultured It was confirmed that GFP colonies were not formed on the plate. Therefore, FACS analysis demonstrated that microbeads containing GFPuv cells can be separated and separated from microbeads not containing GFPuv cells.

(5) MU Containing Microbead Production and FACS analysis

4-methylumbelliferone, a degradation product of 4-methylumbelliferyl-β-D-cellobioside (hereinafter referred to as 'MUG ₂ '), a representative substrate of exo-cellulase 4-Methylumbelliferone (hereinafter referred to as 'MU') to prepare an h / o emulsion. Then, the oil was removed and microbeads were obtained and subjected to fluorescence microscopy and FACS analysis.

LB liquid medium containing 3% (w / v) low melting agarose was sterilized and stored in a 65 ° C incubator. 1 ml of the stored agarose-LDB medium solution was taken in a 1.5 ml microtube and placed in a hit block set at 40 ° C so that the temperature of the solution became 40 ° C.

1 ml of culture broth ( E. coli XL1-blue, OD ₆₀₀ = 0.6) was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed and the cells were washed with 1 × PBS. The cells were resuspended in 1 ml of agarose-LB medium solution containing 1 mM MU at 40 ° C, and 200 μl of the suspension was collected to obtain 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil).

The resulting h / o emulsion was immobilized on a hit block at 40 ° C and homogenized for 1 minute at 9,500 rpm using a homogenizer. The gel was allowed to stand on ice for 5 minutes, and the prepared h / o emulsion was observed under a fluorescence microscope Respectively.

After removing the oil, FACS analysis was performed. For the FACS analysis, a violet laser suitable for the MU wavelength range (Excitation 360 nm, Emission 460 nm) was used.

Fluorescence microscopy revealed that the microbeads were successfully formed and also had strong fluorescence intensity 20 minutes later (Fig. 11).

The FACS analysis also confirmed that the fluorescence intensity was distinctly higher than that of the control (beads containing no MU) and was clearly distinguished (Fig. 12), suggesting that the MU diffusion problem of the existing w / o / w emulsion method was improved And confirmed the feasibility of ultra-high-speed screening of new enzymes using the same.

(6) Exocellulase Expressing cells MUG ₂ Production of h / o using substrate and FACS analysis

By applying the exo-cellulase expression of E. coli (E. coil) cells and MUG substrate ₂ it was produced in the h / o emulsion. A pHSG-celEdx16 plasmid (Appl. Microbiol. Biotechnol. 2011, 89: 1453-1462) containing DNA for expression of exocellulase was transformed into E. coli XL1-blue. Transformed E. coli cells were grown until OD ₆₀₀ = 0.6.

1 ml of culture broth was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed, and the cells were washed with 1 × PBS. The washed cells were resuspended in 1 ml of agrose-LB medium containing 1 mM MUG ₂ , and 200 μl of the suspension was collected to prepare 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil). Fixed at 40 ° C hit block and homogenized for 1 minute at 9,500 rpm using a homogenizer to form an h / o emulsion, which was allowed to stand on ice for 5 minutes to gel. The prepared h / o emulsion was reacted at 37 ° C for 24 hours, and the oil was removed and confirmed by fluorescence microscopy and FACS analysis was performed.

It was confirmed that exocellularase-expressing E. coli cells decompose the MUG ₂ substrate in the microbeads and fluorescence appears in the beads (FIG. 13). Also, FACS analysis showed that the control group (wild type E. coil XL1-blue) (FIG. 14). As shown in FIG.

In addition, wild-type Escherichia coli (E. coil) cells and exo-cellulase expression of E. coli (E. coil) cell a 9: 1 ratio h to mix in the same manner as / o reaction at 37 ℃ for 24 hours after making the microbeads . After removal of the oil, microbeads were obtained and analyzed by FACS and sorted in the order of fluorescence intensity (Fig. 15).

A group having a fluorescence value similar to that of the negative control and a group having a high fluorescence value were each sorted and then spread overnight on an LB agar plate. Each of the resulting colonies was inoculated on a 96-well cell culture plate in LB medium containing 1 mM MUG ₂ , cultured for 12 hours, and then confirmed using a UV transilluminator (Fig. 16).

After transferring 100 쨉 l to a black plate, fluorescence values were measured on a 1420 VICTOR multilabel counter (PerkinElmer Life Sciences, Wallac Finland Oy, Turku, Finland; excitation = 365 nm, emission ≧ 460 nm).

As a result of measurement, it was confirmed that only three out of the 28 colonies obtained by sorting the population with higher fluorescence value than the negative control group were wild-type E. coil cells and all the other cells were exocellulase-expressing cells that degrade MUG ₂ Respectively. Perhaps three false positives were expected to result from the mixing of wild-type cells with exo-cellulase-expressing cells in a single microbead during emulsion formation. In addition, the colonies obtained by sorting the groups showing fluorescence values similar to those of the negative control group were all found to be wild-type E. coli cells.

Therefore, it has been confirmed that beads including positive cells can be successfully detected and separated through FACS. This confirms that the present invention can be effectively used as a tool for ultra-fast screening of new enzymes.

Example 2. Preparation of Poly (ethylene glycol diacrylate) < RTI ID = 0.0 > PEGDA ) Bead IVC H / o production using technology and FACS analysis

The composition of the solution for the polymerization of PEGDA was reviewed by Dr. Lee (Biotechnol Bioeng. 2010, 107 (4): 747-751).

GFP-expressing E. coli cells were grown to OD ₆₀₀ = 0.6. 1 ml of culture broth was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed, and the cells were washed with 1 × PBS. Cells were resuspended in 1 ml of PEGDA solution [0.19% (w / v) and potassium persulfate, 0.6% (w / v) D-sorbitol, 24.8% (w / v) PBS, and 200 μl of the suspension was collected and placed in 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil) containing 0.1% TEMED. Homogenization was performed at 8,000 rpm for 1 minute using a homogenizer to prepare an h / o emulsion, which was then polymerized by incubation at 37 DEG C for 30 minutes. The oil was removed using hexane and then confirmed by fluorescence microscopy and FACS analysis (FIGS. 17 and 18).

Microscopic observations confirmed the successful formation of hydrogel beads, and FACS analysis successfully demonstrated hydrogel beads containing GFP.

Example 3. h / o In the emulsion Effective oil film removal and Microbead Measurement of amount of yield

(1) h / o In the emulsion Cell survival by oil film removal method

1 ml of various concentrations of organic solvents (0, 10, 20, 30, 40, 50%) diluted with 1 x PBS were dispensed into 2 ml microtube. 100 μl of the prepared h / o emulsion is taken, placed in each organic solvent, and vortexed for 10 seconds. Centrifuge at 8,000 rpm for 30 seconds and carefully remove all supernatant. Add 1 ml of PBS, carefully replicate the microbeads, and transfer to a new Eppendorf tube. The microbeads thus formed were spread in an amount of 50 μl each on an LB plate and cultured at 37 ° C. for one day to observe the number of colonies (Table 1).

In addition, we developed a method to remove oil using organic solvent and developed a method to remove oil film using mineral oil.

100 μl of the prepared h / o emulsion is sampled and placed in a 2 ml Eppendorf tube containing 1 ml of mineral oil. After vortexing for 1 minute, centrifugation was performed at 3,000 rpm for 30 seconds. After the oil in the upper layer was removed, 1 ml of mineral oil was again added and the microbeads were washed by vortexing for 1 minute. After centrifuging for 30 seconds, the oil in the upper layer was removed. After the washing step was repeated one more time, the obtained microbeads were resuspended in PBS and centrifuged at 3,000 rpm. The supernatant was carefully removed and resuspended in 1 ml of PBS. 50 μl of the microbeads thus obtained were spread on the LB plate and cultured at 37 ° C. for one day to confirm the number of colonies (Table 1).

Table 1. Colony Survival Rate by Oil Removal Method

The oil removal method using mineral oil was the safest and the highest survival rate for the cells, while the oil removal method using 2-propanol showed the lowest safety. Interestingly, the number of colonies was increased when the oil was removed using hexane compared to the control with only the oil removed with PBS alone. This can be explained by the amount of beads recovered. When hexane was used, the oil was removed smoothly. When the amount of beads was recovered, the yield of the microbeads was increased and the number of colonies was increased .

(2) h / o From the emulsion Depending on oil film removal method Microbead Recovery rate measurement

The recovery rate was measured by comparing the number of microbeads formed during the generation of the h / o emulsion and the number of recovered microbeads after removing the oil using hexane and mineral oil. The number of microbeads when the oil was removed by each method was confirmed using a hemmacytometer (Table 2 and Figure 19).

* A batch method calculates the total number of microbeads per produced; Total number of microbeads / batch (600 μl) = total number of gel beads / 4 × 10 ⁴ × 6

50 μl of the prepared h / o emulsion was taken and added to 950 μl of mineral oil, diluted 20-fold, and then 10 μl was collected and confirmed by a microscope using a hemocytometer (FIG. 19 ).

In addition, 100 mu l of the h / o emulsion was removed with PBS, hexane and mineral oil, respectively, by the oil film removal method described above. The oil-free microbeads were resuspended in final 1 ml of PBS and diluted 10-fold. 10 [mu] l of each of the 10-fold diluted resuspension solutions was collected and confirmed by a microscope using a hemocytometer (Fig. 19). In the case of the h / o emulsion before the oil was removed, the number of beads was calculated by multiplying 2 by the above calculation method since it was diluted twice as much as the oil-removed bead.

The number of microbeads and recovery rates obtained from each oil removal method from the h / o emulsion are shown in the table (Table 2).

Table 2. Number and recovery of microbeads

The total number of h / o microbeads (600 μl emulsion / tube) before oil film removal was about 1.06 × 10 ⁷ . When the oil was removed with 10% hexane using this sample, 8.16 × 10 ⁶ microbeads could be recovered, and 7.56 × 10 ⁶ microbeads could be recovered when mineral oil was used.

Finally, considering the oil film removal method, it was confirmed that the removal method using the mineral oil having the highest microbead yield and the highest cell survival rate is most suitable.

Comparative Example 1. Oil-in-water Of water-in-oil emulsions IVC FACS analysis using

(One) GFP Production and confirmation of w / o / w using cells

In LB broth until the OD ₆₀₀ = 0.6 were grown for (green fluorescent protein) expressing E. coli (E. coil) GFP cells. 1 ml of culture broth was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed, and the cells were washed with 1 × PBS and resuspended in 1 ml of LB liquid medium.

(Nat. Methods 2006, 3: 561-570) to form water-in-oil-in-water w / o / w double emulsions ). 200 [mu] l of the suspended cells were collected and placed in 400 [mu] l of an oil surfactant mixture (2.9% ABIL EM90 in mineral oil). And homogenized for 1 minute at 8,000 rpm at room temperature using a homogenizer (IKA Ultra Turrax T10) to form a w / o emulsion.

The resulting w / o emulsion was added with 500 μl of a buffer solution (assay buffer; 1.5% CMC, 0.5% Triton X-100) and homogenized for 1 minute at 8,000 rpm at room temperature to obtain a w / o / ) Emulsion. The completed emulsion was diluted 20-fold with 1 x PBS and confirmed to be IVC formed w / o / w emulsion successfully containing GFP cells using a fluorescence microscope (Leica) (Fig. 20).

(2) MU Manufacture and confirm w / o / w using phosphor

4-methylumbelliferone, a degradation product of 4-methylumbelliferyl-β-D-cellobioside (hereinafter referred to as 'MUG ₂ '), a representative substrate of exo-cellulase 4-Methylumbelliferone (MU), and IVC was confirmed by fluorescence microscopy. The IVC was carried out to actually apply cells and substrates which express and secrete the cellulase. The production method is as follows.

E. coli until the OD ₆₀₀ = 0.6 (E. coil XL1-blue) cells. 1 ml of culture broth was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed, and the cells were washed with 1 x PBS. The Escherichia coli (E. coil XL1-blue) cells were resuspended in 1 ml of LB liquid medium containing 1 mM MU, and 200 μl of them were collected to prepare 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil). Homogenized for 1 minute at 8,000 rpm using a homogenizer to form a w / o emulsion.

500 μl of assay buffer (1.5% CMC, 0.5% Triton X-100) was added to the w / o emulsion thus formed and homogenized at 8,000 rpm for 1 minute at room temperature using a shredder to obtain w / o / w double emulsion was completed. The completed emulsion was diluted 20-fold with 1 x PBS and confirmed using a fluorescence microscope (Reica) (Fig. 21).

(3) Cellulase expressing cells and MUG ₂ Fabrication and analysis of w / o / w using substrate

Fluorescence signals due to substrate degradation in w / o emulsion were confirmed by applying MUG ₂ substrate and transfected cells expressing the actual cellulase.

Plasmid DNA containing inserts for expression of exocellulase was introduced into XL1-blue E. coil cells and transformed. Transformed E. coli cells are grown to OD ₆₀₀ = 0.6. 1 ml of culture broth was collected and centrifuged at 6,000 rpm for 5 minutes. The supernatant was carefully removed, and the cells were washed with 1 × PBS. The cells were resuspended in 1 ml LB broth containing 1 mM MUG ₂ , and 200 μl of the suspension was collected and added to 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil). And homogenized for 1 minute at 8,000 rpm using a homogenizer (IKA Ultra Turrax T10 homogenizer) to form a w / o emulsion. The formed w / o emulsion was reacted at 37 DEG C for 24 hours and then confirmed by fluorescence microscopy (Fig. 22).

Fluorescence signal due to decomposition of MUG ₂ substrate by cell-expressing cells in the liquid layer in the w / o emulsion was confirmed. However, fluorescence signals were observed in the liquid layer of all emulsions, with or without cells inside the w / o emulsion. Of course, the fluorescence signal in the emulsion containing the cells is a little stronger than the emulsion without the cells. The fluorescence signal in the cell-free emulsion was confirmed to be that the MU, the degradation product of the MUG ₂ substrate, passed through the oil layer and diffused into the other emulsion.

After confirming the above diffusion results, the following diffusion analysis was carried out in order to more clearly confirm the diffusion of MU out of the oil. 200 μl each of the sample with and without 1 mM MU in 100 mM NaCl solution was taken and placed in 400 μl of an oil surfactant mixture (2.9% ABIL EM 90 in mineral oil). Homogenized for 1 minute at 8,000 rpm using a homogenizer to form a w / o emulsion.

500 μl of Assay buffer (assay buffer, 1.5% CMC, 0.5% Triton X-100) was added to the w / o emulsion thus formed and homogenized at 8,000 rpm for 1 minute at room temperature using a homogenizer to obtain w / o / w double emulsion. Each w / o / w emulsion with or without MU was mixed at 1: 1 (v / v) and observed through a microscope (Fig. 23).

Immediately after mixing, fluorescence was observed only in the IVC of the w / o / w double emulsion, but fluorescence signals were observed in all the emulsions even though there was a difference in intensity of the fluorescence signal after 20 minutes. In comparison with the results of optical microscopy, it was confirmed that the IVC form remained, but fluorescence exited outside the IVC.

If the MU does not diffuse through the oil film, the ratio of the emulsion containing MU to the emulsion not containing should be observed at about 1: 1, but since fluorescence signals are observed in all emulsions after 20 minutes, And diffused into all the emulsions.

In addition, the emulsion containing MU was diluted 20-fold with PBS and the fluorescence signal was analyzed by FACS (BD Biosciences, USA). For the FACS analysis, a violet laser suitable for the wavelength range of MU detection (Excitation 360 nm, Emission 460 nm) was used.

Despite the expectation that the fluorescence signal would be detected higher than the control (emulsion without MU), the fluorescence intensity detection pattern was similar to that of the control group (Fig. 24). It was judged that the emulsion was caused by a decrease in fluorescence intensity due to the diffusion of MU after dilution with PBS. The results are also predictable through published papers (Anal Bioanal Chem. 2012, 404 (5): 1439-1447, Anal Chem. 2013, 85: 9807-9814).

In addition, FACS analysis of w / o / w emulsion containing GFP cells did not yield successful results. Although a large difference compared to the control ( E. coli ) was considered, similar analysis results were obtained in which GFP cells were not specifically detected rather than thought (FIG. 25). Although the exact reason is unknown, applying the IVC technique of w / o / w to FACS analysis is considered to be sensitive in many ways. Based on the above results, it was confirmed that the existing w / o / w method as an IVC-based enzyme search technique has sensitivity and limitations in using a substrate that produces a low molecular weight fluorescent product.

Claims

(a) preparing a liquid medium comprising a hydrogelation-forming material;
(b) adding the prepared liquid medium to cells to form a suspension;
(c) adding the suspension of step (b) to an oil surfactant mixture; And
(d) homogenizing and gelating the suspension after step (c) to form a hydrogel-in-oil emulsion containing the cells; and extracellular compartmentalization in vitro compartmentalization (IVC) method.

According to claim 1, wherein the step (d) after the other, the cell according to claim 1, further comprising the step of culturing for 1 to 24 hours at 30 to 37 ℃ the cells contained in the emulsion compartmentalization (in vitro compartmentalization; IVC) method.

The method according to claim 1, wherein in step (b), an extracellular compartmentalization is performed in which a substrate is further added to form a suspension ( in vitro compartmentalization; IVC) method.

The method of claim 1, wherein the hydrogel formation material is selected from the group consisting of agarose, chitosan, alginate, polyethylene glycol, polyvinyl alcohol, polyacrylamide, Polyethylene glycol diacrylate (PEGDA), polyurethane, polypropylene glycol, ployvinylpyrrolidone, xanthan, hyaluronan, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone, wherein at least one selected from the group consisting of gelatin, polyacrylic acid, carboxymethyl celluose and collagen is in an extracellular compartment ( in vitro compartmentalization; IVC) method.

The method of claim 4, wherein the agarose is from 2 to 4% (w / v) compartmentalization extracellular, characterized in that the low melting point agarose (low melting agarose) (in vitro compartmentalization; IVC) method.

The method of claim 1, wherein the homogenization (homoginization) is the extracellular characterized in that the homogenized for 30 to 180 seconds as 8,000 to 13,500rpm, using a homogenizer compartmentalized (in vitro compartmentalization; IVC) method.

The in vitro compartmentalization (IVC) method according to claim 1, wherein the cell is a single cell.

The method of claim 1, wherein the cell is an E. coli extracellular compartmentalization ( in vitro compartmentalization; IVC) method.

A hydrogel-in-oil emulsion containing cells prepared by the extracellular compartmentalization (IVC) method according to any one of claims 1 to 8.

10. The hydrogel-in-oil emulsion according to claim 9, wherein the emulsion is a microbead having an average diameter of 2 to 50 mu m.

(a) adding a substrate to a hydrogel emulsion in an oil containing cells according to claims 9 or 10;
(b) reacting the substrate added in the step (a) with an enzyme contained in cells in the hydrogel; And
(c) confirming the reaction result of the step (b) by a fluorescence-activated cell sorting (FACS) method.

12. The method of claim 11, further comprising the step of removing the oil film from the hydrogel-in-oil emulsion containing cells before the step (a).

13. The method of claim 12, wherein the removal of the oil film comprises using any one selected from the group consisting of mineral oil, hexane, and 2-propanol.