KR101745415B1 - A microfluidic device for capable of screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior and a method for screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior using thereof - Google Patents

A microfluidic device for capable of screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior and a method for screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior using thereof Download PDF

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KR101745415B1
KR101745415B1 KR1020150158843A KR20150158843A KR101745415B1 KR 101745415 B1 KR101745415 B1 KR 101745415B1 KR 1020150158843 A KR1020150158843 A KR 1020150158843A KR 20150158843 A KR20150158843 A KR 20150158843A KR 101745415 B1 KR101745415 B1 KR 101745415B1
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main channel
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심상준
최홍일
김영환
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고려대학교 산학협력단
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Abstract

A microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using chemotaxis and a method of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using chemotaxis are disclosed. A microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability using chemotaxis according to an embodiment of the present invention includes a main channel; A culture medium and a cell inlet connected to one end of the main channel, wherein the suspension containing the first medium in which the organic carbon source is removed, the second medium in which the bicarbonate ions are dissolved and the cells are introduced into the main channel; And a cell outlet connected to the other end of the main channel and through which a first medium, a second medium, and a suspension flow out along the longitudinal direction of the main channel, wherein the first medium, the second medium, And a concentration gradient of bicarbonate ions is formed along the width direction of the main channel in a region adjacent to the culture medium and the cell outflow portion of the main channel.

Description

TECHNICAL FIELD [0001] The present invention relates to a microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using chemotaxis, and a method of selecting a microorganism having excellent carbon dioxide fixing efficiency and growth ability using chemotaxis CARBON DIOXIDE FIXATION EFFICIENCY AND GROWTH RATE BASED ON CHEMOTAXIS BEHAVIOR AND METHOD FOR SCREENING IMPROVED STRAINS WITH HIGH CARBON DIOXIDE FIXATION EFFICIENCY AND GROWTH RATE BASED ON CHEMOTAXIS BEHAVIOR USING THEREOF}

The present invention relates to a microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability using chemotaxis, and a method for selecting a strain having excellent carbon dioxide fixing efficiency and growth ability using chemotaxis, A microfluidic device and a microorganism capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using a chemotaxis capable of rapidly and efficiently selecting a microorganism having a characteristic of reacting to a chemotaxis that reacts with the concentration of The present invention relates to a method for selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using Mars.

In recent years, the problem of exhaustion of energy resources due to the rapid increase of fossil fuel usage and the problem of global warming due to greenhouse gas emissions have attracted worldwide attention. One of the alternatives to solve this problem is microalgae.

Microalgae generally contain chromophores such as chlorophyll, carotenoid, and phycobilin. It refers to single cell organisms that synthesize the organic materials required for cell growth and growth through photosynthesis.

The microalgae are considered to be a real alternative to the above problems because they have a higher growth rate than plants and have the ability to produce large quantities of light energy and neutral lipids capable of converting carbon dioxide and inorganic materials into biodiesel.

To date, several hundred thousand species of microalgae have been reported to exist in freshwater and marine ecosystems and research and development are being tried for various purposes.

However, due to some limitations, there is no way to commercialize them. The biggest problem is the lack of productivity and economy. In order to realize this technology, efforts are being made to solve problems through gene manipulation and cell screening, but the development in this field is still insignificant.

Chlamydomonas Reinhardtii is the most studied microalgae so far. It is considered to be a model organism of microalgae because it is easy to genetically manipulate such as transformation and development of related tools as well as its genome sequence as compared with other strains.

Therefore, it has been actively used for the study of photosynthesis mechanism of microalgae, and it has also been used for lipid-related research and hydrogen production research for biodiesel production.

In the case of photosynthetic organisms, there is an important structure in the photosynthetic activity called thylakoid inside the chloroplast. Especially Chlamydomonas In the microalgae such as reinhardtii , a large amount of carbonic anhydrase is present in the structure to convert the bicarbonate ion present in the water into carbon dioxide, which is a carbon form necessary for photosynthesis, And the ability to increase the efficiency of photosynthesis.

On the other hand, in the case of motile microorganisms such as E. coli , there is a dominant reaction which moves in a certain direction in response to a peripheral stimulus. For example, the main cause induced by light is chemotactic, and the chemotactic main cause induced by chemical is called chemotaxis. Chlamydomonas reinhardtii , a microalgae of movement, is also known to have diverse characteristics.

Especially, the main character which is studied much in microorganisms is the chemotaxis, which means that it detects movement by sensing the type and concentration of the surrounding environmental substance. To date, there have been various studies on the coitalization of Chlamydomonas reinhardtii . However, until now, the research focused mainly on observing the response to various substances such as amino acid and ammonium ion, Related research was insignificant. In particular, there is no known chemotaxis study using bicarbonate ion as a chemical species.

Therefore, in order to increase the productivity of useful materials by improving the CO2 fixation efficiency in the microalgae mass culture system and to contribute to the global warming resolution, it is necessary to determine the coincidental and other cell physiological characteristics of bicarbonate ions, which are closely related to the photosynthesis of microalgae It is necessary to study and understand the characteristics such as carbon dioxide fixing efficiency and growth.

Korean Patent Publication No. 10-2014-0094143 (published on July 30, 2014)

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a screening method capable of quickly and efficiently screening strains having a characteristic of reacting according to the concentration of a carbon source, that is, a reaction characteristic to chemotaxis. In other words, this technology has a correlation with the degree of chemotaxis of microalgae to bicarbonate ions in the microfluidic device in which a stable concentration gradient is formed, the fixing efficiency of carbon dioxide and the growth potential, .

According to an aspect of the present invention, A medium and a cell inlet connected to one end of the main channel, wherein a suspension containing a first medium in which organic carbon source is removed, a second medium in which bicarbonate ions are dissolved and cells are introduced into the main channel; And a culture medium outlet and a cell outlet connected to the other end of the main channel and through which the first medium, the second medium, and the suspension migrate along the longitudinal direction of the main channel, and the first medium and the second medium And the suspension are moved along the longitudinal direction of the main channel to form a laminar flow and a stable concentration gradient of bicarbonate ions along the width direction of the main channel in a region adjacent to the culture medium and the cell outflow portion of the main channel A microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability can be provided by using the characteristic of being formed and the chemotaxis of microalga to detect it.

A first medium inlet connected to one end of the main channel for introducing the first medium and causing the first medium to form a laminar flow along the longitudinal direction of the main channel; A second medium inlet connected to one end of the main channel for introducing the second medium and causing the second medium to form a laminar flow along the longitudinal direction of the main channel; And a suspension inlet connected to one end of the main channel for introducing the suspension and causing the suspension to form a laminar flow along the longitudinal direction of the main channel between the first and second media. have.

Each of the first media inlet, the second media inlet, and the suspension inlet may include a plurality of suspension slits protruded from each other on the inner surface so that the suspension contained in the first medium and the second medium and the suspension may be caught.

The ratio of the outlet side width of the first medium inlet and the second medium inlet adjacent to the main channel to the outlet side width of the suspension inlet adjacent to the main channel may be 10: 1.

Wherein the ratio of the flow rate of the first medium and the second medium flowing into the main channel to the flow rate of the suspension flowing into the main channel from the suspension inlet at the first medium inlet and the second medium inlet is 10: .

The apparatus may further include a pump for supplying the first medium, the second medium, and the suspension to the first medium inlet, the second medium inlet, and the second medium inlet, respectively.

The medium and the cell outlet are connected to the other end of the main channel so that the first medium and the second medium and the suspension moved along the longitudinal direction of the main channel are flowed out, And a plurality of media and a cell outlet through which the cells moving according to a concentration gradient of bicarbonate ions formed along the width direction of the main channel are formed.

Each of the plurality of media and the cell outlets may be formed in a widened configuration in which the inlet side widths adjacent to the main channel are formed to be equal to each other and the width increases toward the outlet side.

Each of the plurality of media and cell outlets may be provided with an opening hole for allowing the first medium, the second medium, and the suspension to flow out to the outside.

The other end of the main channel may be formed outwardly convex so that the inlet side widths of the plurality of media and the cell outlet are formed to be equal to each other.

The second medium can dissolve 100 mM bicarbonate ions.

The main channel, the culture medium, the cell inlet, the culture medium, and the cell outlet may be formed of a light transparent transparent material containing PDMS (polydimethylsiloxane).

According to another aspect of the present invention, there is provided a method of producing a cell, comprising: culturing cells exhibiting a response characteristic to bicarbonate ion; Feeding the first medium, the second medium and the suspension to the medium and the cell inlet of the microfluidic device according to claim 1; And analyzing the behavior of cells according to a concentration gradient of bicarbonate ions formed along a width direction of the main channel in a region adjacent to the culture medium and the cell outflow portion of the microfluidic device, And a method of selecting a strain having excellent growth ability can be provided.

The step of culturing the cells comprises seed culturing the cells and culturing the cells in the first medium for 24 hours at a constant light intensity of 30 μmol photon m -2 s -1 and for 2 days at 23 ° C. have.

When the cells cultured in the first medium collapsed into the exponential phase, cells that were floating in a band-like manner were sensitively reacted with light among the cultured cells, and diluted to a concentration of 1.5 × 10 6 cells ml -1 The method comprising the steps of:

The supplying of the first medium and the second medium and the suspension may include supplying the first medium to a first medium inlet connected to one end of the main channel so that the first medium flows along the longitudinal direction of the main channel, And the second medium is supplied to a second medium inlet connected to one end of the main channel so that the second medium moves in a laminar flow direction along the longitudinal direction of the main channel, The suspension may be supplied to the suspension inlet connected to one end of the channel so that the suspension moves between the first and second media forming a laminar flow along the longitudinal direction of the main channel.

The medium and the cell outlet are connected to the other end of the main channel so that the first medium and the second medium and the suspension moved along the longitudinal direction of the main channel are flowed out, Wherein the analysis of the behavior of the cells comprises a plurality of media and a cell outlet arranged so as to form a path through which cells moving according to a concentration gradient of bicarbonate ions formed along the width direction of the main channel are formed, The number of cells reaching each of the plurality of media and cell outlets can be divided by the total number of cells and normalized before analysis of the viruses.

The present invention can effectively select strains modified using the microfluidic device using the chemotaxis that reacts with the concentration of the carbon source of the photosynthetic single cell organism.

In addition, the present invention facilitates monitoring in a cell unit through a microfluidic device and allows easy selection of an excellent strain against chemical stimulation through various analyzes including statistical analysis of results obtained through a microfluidic device, And it is useful to select strains with excellent growth potential.

FIG. 1 is a schematic diagram illustrating the entire process of selecting strains having excellent carbon dioxide absorption ability by using chemotaxis in the microfluidic device of the photosynthetic microalgae strains according to the present invention.
2 is a photograph of a microfluidic device according to the present invention.
3 is a device diagram showing the structure of the microfluidic device of the present invention and the dimensions of each channel.
FIG. 4 is a graph showing the fluorescence expression photographs over time in the region adjacent to the culture medium and the cell outflow portion of the present invention, and the fluorescence expression level with time of diffusion of 0.1 mM fluorescent substance in the device.
FIG. 5 is a view showing the number of the photosynthetic single-celled organisms reaching the culture medium and the cell outflow of the present invention, in which each inlet and outlet are numbered for statistical analysis.
FIG. 6 is a graph showing statistical analysis of the responses of the wild type strains to different conditions, that is, a condition in which a general culture medium is flowed to both sides and a medium in which a medium having a concentration of bicarbonate of 100 mM is flowed to both sides, That statistic.
FIG. 7 is a graph showing the results of three mutant strains (M1, M2, M3) having a mutagenic property and a change in the chemotactic property to bicarbonate ions by mutation according to the present invention and a culture medium having a normal culture medium and a bicarbonate ion concentration of 100 mM Is the distribution of the outgrowth of photosynthetic single cell organisms observed according to the concentration gradient generated by
FIG. 8 is a graph showing the correlation between numerical expression of chemotaxis, growth, and photosynthesis efficiency of the wild type strain and the three mutant strains used in the present invention.

In order to fully understand the present invention, operational advantages of the present invention, and objects achieved by the practice of the present invention, reference should be made to the accompanying drawings and the accompanying drawings which illustrate preferred embodiments of the present invention.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments of the present invention with reference to the accompanying drawings. Like reference symbols in the drawings denote like elements.

The present invention provides a method for screening mutant strains exhibiting a specific reaction to bicarbonate ion concentration, which is a carbon source, against various mutant strains of photosynthetic single cell organisms having motility in the microfluidic device (100).

The method may comprise the steps of inducing chemotaxis of a single cell organism and yielding a chemotaxis index induced. In addition, the chemotaxis index is obtained by normalizing the number of total cells reached in a plurality of media and cell outlets 151 in a microfluidic device 100 for a predetermined period of time, divided by the total number of cells, Can be analyzed and derived. The chemotaxis used herein refers to the reaction of photosynthetic organisms in response to the concentration of carbon source (or bicarbonate ion) used as an energy source.

To this end, the present invention provides a microfluidic device (100) capable of facilitating observation of individual cells by applying a flow of fluid to quickly and efficiently observe and analyze mobility due to the chemotaxis of the photosynthetic single cell organism.

Referring to FIGS. 1 to 5, a microfluidic device 100 capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability using chemotaxis according to an embodiment of the present invention includes a main channel 110, A first culture medium connected to one end of the channel 110 and containing a second culture medium in which the organic carbon source is removed and a second culture medium in which bicarbonate ions are dissolved and cells are introduced into the main channel 110, And a culture medium outlet 150 connected to the other end of the main channel 110 and through which the first culture medium, the second culture medium, and the suspension are dispensed, which are moved along the longitudinal direction of the main channel 110.

In the microfluidic device 100 according to the present invention, the first medium, the second medium and the suspension form a laminar flow and are moved along the longitudinal direction of the main channel 110, and the medium of the main channel 110 and the cell outlet 150 is formed in a region of the main channel 110 along the width direction of the main channel 110. The strains having excellent carbon dioxide fixing efficiency and growth characteristics are selected.

The main channel 110 serves to diffuse medium carbon ions while the first medium, the second medium and the suspension introduced from the cell inlet 130 flow through the laminar flow to form a concentration gradient.

The main channel 110 has a suitable length and width to have a stable and suitable concentration gradient in the flow conditions of the fluid, preferably 20000 to 400000 mu m in length and 1000 to 2500 mu m in width.

The medium and the cell inlet 130 serve to supply the first medium, the second medium, and the suspension to the main channel 110.

The medium and the cell inlet 130 are connected to one end of the main channel 110. The first medium is a first medium for introducing a first medium and forming a laminar flow along the longitudinal direction of the main channel 110, And a second medium inlet port 133 connected to one end of the main channel 110 for introducing the second medium medium and causing the second medium medium to form a laminar flow along the longitudinal direction of the main channel 110, A suspension inlet connected to one end of the main channel 110 for allowing the suspension to flow and causing the suspension to form a laminar flow along the longitudinal direction of the main channel 110 between the first and second media, ).

The first medium inlet port 131 is disposed above the suspension inlet port 135 and the second medium inlet port 133 is disposed below the suspension inlet port 135. However, The positions of the first medium inlet port 131 and the second medium inlet port 133 can be changed in the upper and lower portions with respect to the first outlet port 135.

The first medium inlet 131, the second medium inlet 133, and the third medium inlet 135 are connected to each other by a plurality of mutually spaced apart protrusions on the inner surface of the first medium, the second medium and the suspension, And a floating suspension block 135a.

The suspension contained in the first medium, the second medium and the suspension is accumulated between the plurality of suspension floats 135a and prevents the suspension from flowing into the main channel 110 by the suspension float 135a. This is to prevent the flow of the fluid in the main channel 110 from being obstructed by the float to change the flowing laminar flow in the main channel 110 to turbulent flow.

The width of the outlet side adjacent to the main channel 110 of the first medium inlet port 131 and the outlet port 133 of the second medium and the width of the main inlet 110 of the suspension inlet 135 are set so that the flow of fluid in the main channel 110 forms a laminar flow. The ratio of the width on the outlet side adjacent to the channel 110 is 10: 1. Specifically, in this embodiment, the outlet side width of the first medium inlet port 131 and the second outlet port 133 is 800 占 퐉, and the outlet side width of the suspension inlet port 135 is 80 占 퐉.

The flow rate of the first medium and the second medium flowing into the main channel 110 from the first medium inlet port 131 and the second medium inlet port 133 and the flow rate of the second medium from the suspension inlet 135 to the main channel 110 Gt; 10: 1 < / RTI > Specifically, in this embodiment, the flow rate of the first medium supplied to the first medium inlet port 131 is 0.4320 μl / min, the flow rate of the first medium supplied to the second medium inlet port 133 is 0.4320 μl / min, And the flow rate of the suspension supplied to the suspension inlet 135 is 0.0432 ㎕ / min.

The microdevice of the present invention comprises a pump (not shown) for supplying the first medium, the second medium and the suspension to the first medium inlet 131, the second medium inlet 133, and the suspension inlet 135, ).

For example, in the present embodiment, a pump may be a syringe, a syringe pump, or the like, and a syringe pump may be used to feed the first medium to the inlet side of the first medium inlet port 131 and the first medium to the inlet side of the second medium inlet port 133 And a suspension is continuously supplied to the inlet side of the suspension inlet 135 by a predetermined amount so that a laminar flow is formed in the main channel 110.

The medium and the cell outlet 150 according to the present embodiment serve to discharge the first medium, the second medium, and the suspension moved along the main channel 110.

The medium and the cell outlet 150 are connected to the other end of the main channel 110 to discharge the first medium and the second medium and the suspension moved along the longitudinal direction of the main channel 110, And a plurality of cells and a cell outlet 151 arranged to be spaced apart from each other along the width direction and forming a path through which cells moving according to a concentration gradient of bicarbonate ions formed along the width direction of the main channel 110 are moved.

In this embodiment, nine media and cell outlets 151 are formed so as to accurately grasp the distribution of cells generated by the host chemotaxis under the flow conditions of the first medium, the second medium, and the suspension flowing through the main channel 110 However, the scope of rights of the present invention is not limited thereto.

Each of the plurality of media and cell outlets 151 is formed to have the same width at the inlet side adjacent to the main channel 110 and to have an enlarged width at the outlet side.

At this time, the other end of the main channel 110, that is, a region to which the plurality of media and the cell outlet 151 are connected, is convex outward so that the inlet side widths of the plurality of media and the cell outlets 151 are formed to be equal to each other . The inlet side widths of the plurality of media and the cell outlet 151 have the same size so that the same flow rate flows into the plurality of media and the cell outlet 151.

In each of the plurality of media and the cell outlets 151, an opening hole (not shown) is formed on the outlet side for allowing the first medium, the second medium and the suspension to flow out to the outside.

Thus, the first medium, the second medium, and the suspension are introduced into the plurality of media and cell outlets 151 through the main channel 110, and then discharged to the outside through the opening holes.

Meanwhile, the main channel 110, the medium and the cell inlet 130, and the medium and the cell outlet are light-transparent and transparent materials, and are not toxic to cells such as microalgae, A material that does not disturb movement or a material that is pretreated to have the above characteristics.

For example, the main channel 110, the medium and the cell inlet 130, and the medium and the cell outlet 150 may be formed of a transparent transparent material including polydimethylsiloxane (PDMS).

As described above, the microfluidic device 100 according to the present invention is formed of a transparent material having transparency of polydimethylsiloxane (PDMS), which is a polymer material, and PDMS has no toxicity to living organisms, PDMS can be custom designed for microfluidic devices suitable for the object and purpose to be analyzed.In particular, based on high transparency and microstructure, PDMS can be applied to individual cells through optical microscope , It is possible to contribute to statistically and precisely understand the movement of the photosynthetic single cell organism according to the chemotaxis.

In addition, since the microfluidic device 100 according to the present invention can miniaturize the analysis system, sample and expensive biochemical reagents can be reduced, and the analysis can be performed simultaneously in parallel, thereby reducing the analysis time and expense . The flow of uniform laminar flow without turbulence also simplifies the experimental conditions and minimizes external influences. In addition, reproducible analytical data can be obtained. In addition, cell-based analysis data have been widely used for drug screening, pharmacokinetics, and toxicity analysis, but they can be used to exchange metabolites or identify intracellular mechanisms between cells and tissues.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to the following examples.

In order to solve the above problems, the present invention provides a microfluidic device (100) in which a flow of a photosynthetic single cell organism is moved by flow and a concentration gradient of bicarbonate ions is formed through diffusion, The present invention provides a method for screening a mutant strain showing an immune response.

At this time, the method includes the steps of making the structure of the microfluidic device 100, fabricating the microfluidic device 100 using polydimethylsiloxane (PDMS), and making a concentration gradient of the carbonic acid ions necessary for photosynthesis (100).

In order to increase the reactivity of the chemotaxis, the photosynthetic single-celled organism is continuously cultured in a medium from which the carbon source has been removed, the photosynthetic organism is put into the medium of the microfluidic device 100 and the cell inlet 130, Counting the number of cells passing through the nine outlets and the cell outlets 151 for a certain period of time, collecting the nine out of the nine outlets and the organisms from the outlets 151, . ≪ / RTI >

The method according to the present invention involves measuring the chemotaxis of wild type and mutant single cell organisms, which can be calculated by statistical analysis of the number of cells passing through nine media and cell outlets 151 for a certain period of time. These indicators include, but are not limited to, analysis of the number of cells moving according to the flow of the fluid, analysis of the kurtosis and the degree of the graph based on the analysis of the pattern of the number of cells reaching the outlet according to the carbon source concentration It is not.

The method according to the present invention may further include a step of analyzing the photosynthetic efficiency and growth potential of the wild type and mutant single cell organisms and statistically analyzing the correlation between these data and the chemotaxis. The method for measuring the photosynthetic efficiency and growth ability may include various indexes, including, but not limited to, a change in photosynthetic pigment, a change in photosynthetic mechanism, and a light conversion efficiency.

The present invention also provides a mutant strain of a photosynthetic single-celled organism which exhibits a specific reaction to bicarbonate ions more easily than a conventional method of isolating strains by biochemical analysis or the like, in a microfluidic device (100) The number of cells passing through the outlet 151 can be represented by a histogram, and an excellent strain can be selected through analysis of the degree of kurtosis and the degree of kurtosis, which is one of the statistical analysis methods.

Particularly, it is possible to accurately analyze microorganism strains which specifically react with bicarbonate ions on an optical microscope through the microfluidic device 100, which is easy to observe the individual reactions of the cells, and can perform statistical analysis However, it does not limit the monitoring on the optical microscope only.

In addition, the present invention can be applied to various indicators (histograms of kurtosis and distortion analysis) obtained by statistical analysis on individual reactivity to carbon sources of photosynthetic single cell organisms, which can indicate the reaction characteristics by chemotaxis and the degree of carbon dioxide fixing efficiency In addition, when there is denaturation of mechanisms and factors related to the ability of CO2 fixation caused by mutation, it is easier to search for the change through simple statistical analysis than complex biochemical analysis, and furthermore, Can be effectively selected.

In addition, the selection of strains that are capable of absorbing the carbon dioxide of mutant strains using the assay including the growth assay of the mutant strains proves the correlation between coinage and growth and photosynthetic efficiency, thereby contributing to the selection of excellent strains at high speed Is possible.

Example  1. Preparation of mutant strains with various reaction characteristics for bicarbonate ion

The strain used in the present invention is Chlamydomonas The wild type strain used for producing the mutant strain by renihardtii is CC125, and from this wild type strain, Chlamydomonas Reinhardtii transformants were transformed by electroporation to obtain mutant strains and various mutant strains were tested for their ability to react with bicarbonate ions in the microfluidic device 100 And analyzed.

The first medium used in this experiment was TAP-C medium with organic carbon source removed, and the constituents of the first medium were shown in Table 1 below.

TAP-C Medium ingredient Content (in 1L water) TAP salts (in 1 L water) 25 ml NH 4 Cl 15.0 g
MgSO 4 · 7H0 4.0 g
CaCl 2 .2H 2 O 2.0 g
Phosphate solution (in 100 ml water) 0.375 ml K 2 HPO 4 28.8 g
KH 2 PO 4 14.4 g
Hutners trace elements 1.0 mL EDTA disodium salt 50 g (250 ml of water)
ZnSO 4揃 7H 2 O 22 g (100 ml water)
H 3 BO 3 11.4 g (200 ml of water)
MnCl 2 .4H 2 O (5.06 g, 50 ml water)
1.61 g of CoCl 2 .6H 2 O (50 ml of water)
CuSO 4 · 5H 2 O 1.57 g (50 ml water)
(NH 4 ) 6 Mo 7 O 24 .4H 2 O 1.10 g (50 ml water)
4.99 g of FeSO 4 .7H 2 O (50 ml of water)
Tris base 2.42 g HCl 1.3 ml

Example  2. Microalgae culture and chemotaxis based selection of microfluidic device

Chlamydomonas Reinhardtii wild-type strain (CC125) and the mutant strain were cultured on TAP agar medium in seed culture at 30 μmol photon m -2 s -1 for 24 hours and 23 Lt; 0 > C for 2 days. This is an adaptation process for increasing the chemical reactivity to the carbon source and for controlling it efficiently and constantly.

After 2 days of incubation, the cells were sensitized by light when they reached the exponential phage stage. Cells that rose in a band shape along the light were collected and diluted to a certain cell concentration (1.5 × 10 6 cells ml -1 ) Used in experiments. This is a process for selecting cells that are active in photosynthesis and movement.

The microfluidic device 100 used in the present invention was manufactured by spin coating a negative photosensitizer SU-850 on a silicon substrate, covering the designed mask, and exposing the mask to ultraviolet rays using photolithography using an ultraviolet exposure machine , Polymer PDMS (Polydimethylsiloxane) and curing agent were mixed at a ratio of 10: 1, and the polymer was poured on a SU-8 mold manufactured by photolithography and then thermally cured. The completed PDMS microfluidic device 100 was bonded to the slide glass (G) through oxygen plasma treatment.

Example  3. Driving the device using a syringe and a syringe pump

In FIG. 3, the first medium, which is a general TAP-C medium, and the third medium, which is a TAP-C solution in which 100 mM of bicarbonate ions are dissolved, are injected into the first medium inlet 131 using a syringe and a syringe pump Inject. At the same time, a suspension containing cells is injected into the suspension inlet 135. After about 15 minutes after the first medium, the second medium and the suspension are moved along the main channel 110, the main channel 110 and the cell outlet 150 are connected to the main channel 110 in a region adjacent to the medium and the cell outlet 150, The number of cells passing through the plurality of media and the cell outlets 151 was counted after a density gradient of bicarbonate ions was stably formed along the width direction of the cell 110.

Example  4. In the microfluidic device Fluorescence Factor  Using constant and continuous Density zone Check ship

4, in order to confirm whether the concentration gradient of the carbon source in the microfluidic device 100 is constant and maintained, it is preferable to use a fluorescence substance, fluorescein having a diffusion behavior similar to that of bicarbonate ions, Fluorescence intensities were measured in the medium of the main channel 110 and in the area adjacent to the cell outlet 150. As a result, compared to the case where the flow of the first medium, the second medium and the suspension is initially formed in the main channel 110, the fluorescent material diffuses and the width of the main channel 110 It is confirmed that a continuous and stable concentration gradient is formed in the direction of the arrow.

These results show that the concentration gradient of the carbon source is maintained constant in the microfluidic device 100 even after a certain time after the concentration gradient is stably formed. Therefore, the carbon source concentration of the photosynthetic single cell organism It is possible to analyze the reaction characteristics according to the gradient.

Example  5. Demonstration of the presence of chemotaxis of photosynthetic single cell organisms on bicarbonate ions through differences in the behavior of wild-type strains at low and high concentrations

In FIG. 6, the behavior of cells in the wild type strain CC125 was observed in the other two environments.

4, when the TAP-C medium (first medium) is supplied to the first medium inlet 131 and the second medium inlet 133 and the cell suspension is supplied through the suspension inlet 135, The distribution of the cells passed through all the media and the cell outlets 151 for 15 minutes after the development, but the TAP When the C (second medium) was supplied, most of the cells were concentrated in the outlets of the plurality of media and the cell outlet 151 at the outlet No. 5.

As shown in FIG. 6, when the histogram of the cell distribution is shown in FIG. 6, the kurtosis indicating mathematical sharpness of the graph is about 0.061 and the kurtosis of the latter is about 2.62. This can be interpreted as a result of the chemotaxis of the cells to avoid excessively high concentration of bicarbonate ion solution and it has been experimentally confirmed that the cells exhibit behavior based on the chemotaxis of bicarbonate ions.

Example  6. Analysis of chemotactic behavior of wild-type and mutant strains

The wild type strain, which was used to select a strain having excellent carbon dioxide fixing efficiency and growth ability through the concentration gradient of bicarbonate ion, which is one of the photosynthetic materials of photosynthetic single cell organisms formed in the microfluidic device 100, and total 3 The number of cells passing through the cell outlets 151 was counted for the mutant strains M1, M2, and M3 of the species, and the skewness was calculated by statistical analysis. The values were compared.

(TAP-C) medium (first medium) was placed in the first medium inlet port 131 and TAP-C (second medium inlet port) 133 having a concentration of 100 mM in the second medium inlet port 133, C solution (second medium) was supplied and the cell culture solution was supplied through the suspension inlet 135. After passing through each of the nine media and cell outlets 151 for 15 minutes after the concentration gradient of the bicarbonate ion is stably formed (about 15 minutes after the first medium and the second medium and the suspension are injected into the main channel 110) The number of cells was calculated. Based on this, the distribution of the cells could be drawn with a histogram (see FIG. 7) and the degree of distortion of the graph was analyzed. Mathematically, the quantity distortion is calculated when the graph is tilted to the left with respect to the mean and vice versa. Therefore, it is obvious that the graph with a degree of zero is symmetrical. Based on this analysis, the cell distribution of mutant strain 1 (M1), wild type strain (CC125), mutant strain 2 (M2) and mutant strain 3 (M3) was analyzed to be 0.475, 0.321, 0.019 and 0.013 respectively. From the physical analysis, it can be seen that M1 is preferred to wild type, M2, and M3 in order of relatively high concentration of bicarbonate ion, and it is confirmed that each strain has different chemotactic behavior.

Example  7. Analysis of correlation between growth and photosynthesis efficiency and chemotaxis index

The correlation between the indicators of the chemotaxis analyzed based on the microfluidic device 100 of the present invention and the growth and photosynthetic efficiency of the strain was analyzed. First, growth characteristics of wild type strain (CC125) and mutant strains M1, M2 and M3 were analyzed. For the growth assay, initial inoculation concentrations were inoculated into TAP-C medium (first medium) at a concentration of optical density (OD) of 0.1 at a wavelength of 800 nm using the absorbance method, and cultured for 6 days And the growth was observed using the difference between the maximum absorbance and the minimum absorbance. As a result, the mutant strain 3 (M3) showed about two times more growth than the control strain wild type strain. Next, the efficiency of intracellular photosystem was measured to show photosynthetic efficiency. Higher photosystem efficiency means higher photosynthetic efficiency, and lower photosystem efficiency means less photosynthetic efficiency.

Based on the measured data, as shown in FIG. 8 (a), the mathematical index of the chemotaxis, that is, the growth property of the tsuki cell, was analyzed. As a result, a high correlation of R 2 = 0.9204 was obtained, and it was found that the linearity was visually high. This indicates that the strains having excellent chemotaxis of the photosynthetic single cell organism have a high growth potential.

Next, as shown in FIG. 8 (b), the correlation between the coincidence and the luminous efficacy was analyzed, showing a very high correlation of R 2 = 0.9675. This means that the chemotaxis of photosynthetic single cell organisms is closely related to the photosynthetic efficiency of the strain.

Finally, as shown in FIG. 8 (c), the relationship between the growth rate and the light efficiency is highly correlated with R 2 = 0.8856, which is closely related to each other.

As a result, it was confirmed that the microfluidic device 100 of the present invention can easily select strains having excellent growth and photosynthetic efficiency through the screening method and the possibility of selecting excellent strains among various mutant strains Respectively.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Accordingly, such modifications or variations are intended to fall within the scope of the appended claims.

100: microfluidic device 110: main channel
130: medium and cell inlet 131: first medium inlet
133: Second medium inlet port 135: Suspension inlet port
150: medium and cell outlet 151: medium and cell outlet

Claims (17)

Main channel;
A medium and a cell inlet connected to one end of the main channel, wherein a suspension containing a first medium in which organic carbon source is removed, a second medium in which bicarbonate ions are dissolved and cells are introduced into the main channel; And
And a culture medium outlet and a cell outlet connected to the other end of the main channel and through which the first medium, the second medium and the suspension migrate along the longitudinal direction of the main channel,
The first medium, the second medium and the suspension form a laminar flow and are moved along the longitudinal direction of the main channel. The first medium, the second medium, and the suspension are dispersed along the width direction of the main channel, The microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using the chemotaxis in which concentration gradient of bicarbonate ion is formed. The method according to claim 1,
The culture medium and the cell-
A first medium inlet connected to one end of the main channel for introducing the first medium and causing the first medium to form a laminar flow along the longitudinal direction of the main channel;
A second medium inlet connected to one end of the main channel for introducing the second medium and causing the second medium to form a laminar flow along the longitudinal direction of the main channel; And
And a suspension inlet connected to one end of the main channel for introducing the suspension and causing the suspension to form a laminar flow along the longitudinal direction of the main channel between the first medium and the second medium, The microfluidic device is capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth property by using the microfluidic device. 3. The method of claim 2,
The first media inlet, the second media inlet, and the suspension inlet, respectively,
A microorganism capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using the chemotactic property including a plurality of floating suspension protrusions which are mutually spaced apart and projected on the inner surface so that the suspension contained in the first medium, the second medium, Fluid device. 3. The method of claim 2,
The ratio of the outlet side width of the first medium inlet and the second medium inlet adjacent to the main channel to the outlet side width of the suspension inlet adjacent to the main channel is 10: 1, and the carbon dioxide fixing efficiency and the growth A microfluidic device capable of selecting this excellent strain. 5. The method of claim 4,
Wherein the ratio of the flow rate of each of the first medium and the second medium flowing into the main channel at the first medium inlet and the second medium inlet to the flow rate of the suspension flowing into the main channel at the suspension inlet is 10: A microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using a chemotaxis of the present invention. 3. The method of claim 2,
And a pump for supplying the first medium and the second medium and the suspension to the first medium inlet, the second medium inlet, and the second medium inlet, respectively, so as to select a strain having excellent carbon dioxide fixing efficiency and growth performance The microfluidic device. The method according to claim 1,
The culture medium and the cell outflow portion may contain,
The first and second media and the suspension, which are connected to the other end of the main channel and are moved along the longitudinal direction of the main channel, are separated from each other along the width direction of the main channel, A microorganism capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using a chemotaxis including a plurality of media and a cell outlet forming a path through which cells moving according to a concentration gradient of bicarbonate ions formed along the width direction Fluid device. 8. The method of claim 7,
Wherein each of said plurality of media and cell outlets comprises:
A microfluidic device capable of selecting a microorganism having excellent carbon dioxide fixing efficiency and growth ability by using a coagulant property formed such that the width on the inlet side adjacent to the main channel is equal to the width on the exit side. 9. The method of claim 8,
In each of the plurality of media and cell outlets,
The microfluidic device is capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using the coagulability in which the first medium, the second medium, and the opening are formed at the outlet side to allow the suspension to flow out. 9. The method of claim 8,
And the other end of the main channel is formed into a microstructure capable of selecting a culture strain having excellent carbon dioxide fixing efficiency and growth property by using the chemotaxis formed outwardly convex so that the inlet widths of the plurality of media and the cell outlets are formed to be equal to each other Fluid device. The method according to claim 1,
Wherein the second medium is capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth ability by using a chemotaxis in which 100 mM bicarbonate ions are dissolved. The method according to claim 1,
The main channel, the medium and the cell inlet, and the medium and the cell outlet,
A microfluidic device capable of selecting a strain having excellent carbon dioxide fixing efficiency and growth property by using a chemotaxis formed of a transparent transparent material containing PDMS (polydimethylsiloxane). Culturing cells exhibiting reaction characteristics for bicarbonate ions;
Feeding the first medium, the second medium and the suspension to the medium and the cell inlet of the microfluidic device according to claim 1; And
Analyzing the behavior of cells in a concentration gradient of bicarbonate ions formed along the width direction of the main channel in a region adjacent to the culture medium and the cell outflow portion of the microfluidic device, Selection method of strains with excellent growth potential. 13. The method of claim 12,
The step of culturing the cells comprises:
Cells were seeded and cultured for 24 h at 30 μmol photon m -2 s -1 in the first medium and chemotaxis for 2 days at 23 ° C. Selection method of strains with excellent growth potential. 15. The method of claim 14,
When the cells cultured in the first medium collapsed into the exponential phase, cells that were floating in a band-like manner were sensitively reacted with light among the cultured cells, and diluted to a concentration of 1.5 × 10 6 cells ml -1 The method comprising the steps of: a. 14. The method of claim 13,
Wherein the step of supplying the first medium, the second medium and the suspension comprises:
Supplying the first medium to a first medium inlet port connected to one end of the main channel to allow the first medium to be moved in a laminar flow direction along the longitudinal direction of the main channel,
Supplying the second medium to a second medium inlet connected to one end of the main channel so that the second medium moves in a laminar flow direction along the longitudinal direction of the main channel,
The suspension is supplied to a suspension inlet connected to one end of the main channel so that the suspension is moved between the first and second media to form a laminar flow along the longitudinal direction of the main channel, A method of selecting strains having excellent fixing efficiency and growth ability. 14. The method of claim 13,
The culture medium and the cell outflow portion may contain,
The first and second media and the suspension, which are connected to the other end of the main channel and are moved along the longitudinal direction of the main channel, are separated from each other along the width direction of the main channel, And a plurality of cells and a cell outlet which form a path through which cells that move according to a concentration gradient of bicarbonate ions formed along the width direction are formed,
Analyzing the behavior of the cells,
A method for screening a strain having excellent carbon dioxide fixation efficiency and growth ability by using the chemotaxis of normalizing the number of cells reaching each of the plurality of media and cell outlets divided by the total number of cells and analyzing the degree of distortion.
KR1020150158843A 2014-11-12 2015-11-12 A microfluidic device for capable of screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior and a method for screening improved strains with high carbon dioxide fixation efficiency and growth rate based on chemotaxis behavior using thereof KR101745415B1 (en)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Anal Bioanal Chem., Vol. 405, No. 29, pp. 9365-9374 (2013.11.)
Lab Chip., Vol. 14, No. 8, pp. 1415-1425 (2014.04.)
Lab Chip., Vol. 15, No. 18, pp. 3687-3694 (2015.08.06.)
Lab Chip., Vol. 7, No. 10, pp. 1371-1373 (2007.10.)

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