Culture vessel, method for static culture of microorganisms, and method for producing proteins

Abstract

To provide a culture vessel for efficiently culturing microorganisms under static conditions. [Solution] A culture vessel for static culture of microorganisms. The culture vessel has multiple culture sections, each having an oxygen permeable layer, and each of the oxygen permeable layers has an oxygen permeability of 150 cm³ measured at 37°C and 0% humidity. 3 / (m 2 ·24h・atm) or more than 90000cm 3 / (m 2 (24 hours / atm) or less.

Description

Technical Field

[0001] The present invention relates to a culture vessel, a method for statically culturing microorganisms, and a method for producing proteins.

Background Art

[0002] The cultivation of microorganisms is carried out, for example, as described in Patent Documents 1 to 4, by shaking a flask containing the microorganisms to be cultivated and a culture medium. By shaking the flask, oxygen can be easily dissolved in the culture medium, or the culture medium can be uniformly mixed to uniformly grow the microorganisms.

[0003] On the other hand, Patent Document 5 describes a method for statically culturing microorganisms having cellulose production ability in order to reduce the influence on flavor in vinegar fermentation, the generation of by-products and the decrease in yield due to the oxidation reaction of glycol in a culture medium having glucose as a main carbon source, and the damage to microorganisms caused by stirring. In Patent Document 5, in order to compensate for the decrease in the oxygen supply amount in static cultivation, the bottom surface portion of a cylindrical container is formed of an oxygen-permeable hydrophobic material, and a configuration is adopted in which oxygen can be supplied to the culture solution on the bottom surface.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0005] As described in Patent Documents 1 to 5, microbial culture has traditionally been carried out using relatively large-capacity containers such as flasks or containers of similar size. However, these methods only allow for the cultivation of microorganisms in one container at a time, making it difficult to improve the efficiency of the culture.

[0006] In recent years, the cultivation of microorganisms on a microscale using microplates has also been investigated. However, culturing microorganisms using microplates requires high-speed rotation, such as 2000 rpm to 3000 rpm for a 384-well plate. Such powerful shaking requires specialized equipment, and there is also a risk of contamination from splashing during shaking.

[0007] The present invention has been made in view of the above problems, and aims to provide a culture vessel for efficiently statically culturing microorganisms, a method for statically culturing microorganisms using the culture vessel, and a method for producing proteins using the culture vessel. [Means for solving the problem]

[0008] One aspect of the present invention for solving the above problems relates to the culture vessels described in [1] to

[14] below, a method for static culture of microorganisms, and a method for producing proteins. [1] A culture vessel for static culture of microorganisms, Each has multiple culture sections, each with an oxygen-permeable layer. All of the aforementioned oxygen permeable layers measured at 37°C and 0% humidity and showed an oxygen permeability of 150 cm³. 3 / (m 2 ·24h·atm) or more than 90000cm 3 / (m 2 (24 hours / atm) or less. Culture container. [2] The oxygen permeable layer comprises a 4-methyl-1-pentene polymer, [1] The culture vessel described above. [3] The 4-methyl-1-pentene-based polymer is at least one polymer selected from the group consisting of a homopolymer of 4-methyl-1-pentene and a copolymer of 4-methyl-1-pentene and ethylene and an α-olefin having 3 or more and 20 or less carbon atoms (excluding 4-methyl-1-pentene). The culture vessel according to [2]. [4] Having 5 or more of the culture parts. The culture vessel according to any one of [1] to [3]. [5] The culture part has the oxygen permeable layer as a bottom member. The culture vessel according to any one of [1] to [4]. [6] The area of the oxygen permeable layer constituting the bottom member when the culture vessel is viewed in plan is 8 mm 2 or more and 960 mm 2 or less. The culture vessel according to [5]. [7] The ratio of the area [mm 2 of the oxygen permeable layer constituting the bottom member when the culture vessel is viewed in plan to the depth [mm] of the culture part is 0.40 mm or more and 230 mm or less. The culture vessel according to [5] or [6]. [8] The ratio of the area [mm 3 of the oxygen permeable layer constituting the bottom member when the culture vessel is viewed in plan to the volume [mm 2 of the culture part is 0.01 mm -1 or more and 0.25 mm -1 or less. The culture vessel according to any one of [5] to [7]. [9] The microorganism is at least one selected from the group consisting of bacteria, archaea, fungi, protists, viruses, and microalgae. The culture vessel according to any one of [1] to [8].

[10] A step of preparing the culture vessel according to any one of [1] to [9], and a step of statically culturing a microorganism inside the culture part of the culture vessel. A method for statically culturing a microorganism.

[11] The culture section has the oxygen permeable layer as a bottom member, In the static culture step, the area of ​​the oxygen permeable layer having as the bottom member is given by the liquid height [mm] of the culture medium containing the microorganisms. 2 The ratio of ] is between 0.40 mm and 230 mm.

[10] A method for static culture of microorganisms.

[12] The culture section has the oxygen permeable layer as a bottom member, In the static culture step, the volume of the culture medium containing the microorganisms [mm²] 3 The area of ​​the oxygen permeable layer having as the bottom member [mm²] 2 The ratio of ] is 0.05 mm -1 0.24mm -1 The following is: A method for static culture of microorganisms as described in

[10] or

[11] .

[13] A step of processing the contents of the culture section after the step of static culture, A method for static culture of microorganisms as described in any of

[10] to

[12] . The process of preparing a culture vessel as described in any of

[14] [1] to [9], The process includes the step of statically culturing microorganisms inside the culture section of the culture vessel, A method for producing proteins. [Effects of the Invention]

[0009] The present invention provides a culture vessel for efficiently culturing microorganisms under static conditions, a method for culturing microorganisms under static conditions using the culture vessel, and a method for producing proteins using the culture vessel. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a perspective view of the culture vessel. [Figure 2] Figure 2 is a cross-sectional view of a portion of the culture vessel. [Figure 3]Figures 3A and 3B are graphs showing the average absorbance measured from each well for each culture condition in Experiment 1 of the Examples. [Figure 4] Figure 4 is a graph showing the mean and standard deviation (SD) of OD600 measured for the 4MP1 plate and PS plate, respectively, in Experiment 2 of the Examples. [Figure 5] Figure 5 is a graph showing the average OD600 values ​​measured for the 4MP1 plate and the LUMOX plate, respectively, in Experiment 3 of the Examples. [Figure 6] Figure 6 is a graph showing the average OD600 values ​​measured for each of the 4MP1-6 plate, 4MP1-24 plate, 4MP1-96 plate, and 4MP1-384 plate in Experiment 4 of the Examples. [Figure 7] Figure 7 is a graph showing the average OD600 values ​​measured for each well of 4MP1-384 plate-1 / 2, 4MP1-384 plate-3 / 4, and 4MP1-384 plate-total volume in Experiment 5 of the Examples. [Figure 8] Figure 8 shows the results of SDS-PAGE performed in Experiment 6 of the Examples. [Modes for carrying out the invention]

[0011] 1.Culture container A first embodiment of the present invention relates to a culture vessel for static culture of microorganisms. In this specification, "culture of microorganisms" means to increase, grow, or maintain a viable state of the microorganisms. "Static culture" means to culture fine materials without applying artificial shaking, such as with a microplate shaker.

[0012] Figure 1 is a perspective view of culture vessel 100, which is a culture vessel for static cultivation of microorganisms. Figure 2 is a cross-sectional view of a part of culture vessel 100.

[0013] The culture vessel 100 is used, for example, to culture prokaryotes such as Escherichia coli. The culture vessel 100 has a plurality of containment sections 210 for culturing microorganisms (see Figure 2; the containment section 210 is an example of a culture section). The culture vessel 100 is placed in the culture space of a culture device (e.g., a constant temperature chamber) with the liquid culture medium and the microorganism to be cultured (hereinafter referred to as "target microorganism") contained in the containment sections 210. However, the culture vessel 100 does not need to be used while placed in a culture device. The culture vessel 100 may be used in various situations depending on the target microorganism.

[0014] The base material 200 has a frame 220 and a resin sheet 230.

[0015] The frame 220 has a frame portion 222 and a plurality of wells 224. The frame 220 is made of, for example, a thermoplastic resin and is an integrally molded product made by injection molding. Examples of thermoplastic resins that make up the frame 220 include polystyrene and polyolefins. Examples of polyolefins include polyethylene, polypropylene, 4-methyl-1-pentene (co)polymers, and cyclic olefin (co)polymers. Of these, 4-methyl-1-pentene polymers are preferred from the viewpoint of improving the gas permeability of the frame 220. Alternatively, 4-methyl-1-pentene polymers, polystyrene, polypropylene, and cyclic olefin polymers are preferred from the viewpoint of improving the heat resistance of the frame 220.

[0016] The frame portion 222 is composed of rectangular frame-shaped members. The well 224 is provided in the space enclosed by the frame portion 222. The well 224 is provided integrally with the frame portion 222.

[0017] The well 224 is cylindrical with an upward opening. In this embodiment, the shape of the opening of the well 224 (in other words, the outer shape in plan view) is circular. The wells 224 are arranged in the left-right and front-back directions. The number of wells 224 (and in the housing section 210) is preferably 5 or more, more preferably 80 or more, and even more preferably 300 or more. For example, the number of wells 224 (and in the housing section 210) may be 6, 24, 96, or 384. Of course, the number of wells 224 may be other than 6, 24, 96, and 384.

[0018] Adjacent wells 224 are connected to each other via connecting parts. In addition, the leftmost well 224, the rightmost well 224, the frontmost well 224, and the rearmost well 224 are connected to the inner circumferential surface of the frame portion 222 via connecting parts.

[0019] The resin sheet 230 is attached to the lower surface of the frame 220 via an adhesive layer 240. This allows the resin sheet 230 to close the bottom of the wells 224, forming the bottom of the culture container 100. In this embodiment, the resin sheet 230 is made of a material with high oxygen permeability. Therefore, the resin sheet 230 also functions as an oxygen-permeable layer, supplying oxygen to the microorganisms cultured inside the containment section 210. In this embodiment, the resin sheet 230 closes the bottom of multiple wells 224. This forms multiple containment sections 210, each having an oxygen-permeable layer.

[0020] The resin sheet 230 is preferably a transparent resin sheet with high oxygen permeability. Examples of such transparent resins include polyethylene, polypropylene, polyethylene terephthalate, polystyrene, polydimethylsiloxane, and 4-methyl-1-pentene (co)polymers.

[0021] Furthermore, it is preferable that the resin used for the resin sheet 230 is a different thermoplastic resin from the thermoplastic resin used for the frame 220. For example, the thermoplastic resin used for the frame 220 can be polystyrene or a cyclic olefin-based (co)polymer, and the resin used for the resin sheet 230 can be a 4-methyl-1-pentene-based (co)polymer. Alternatively, the thermoplastic resin used for the frame 220 can be a 4-methyl-1-pentene-based (co)polymer, and the resin used for the resin sheet 230 can be a 4-methyl-1-pentene-based (co)polymer with different comonomer species or their ratios. By using different thermoplastic resins for these materials, different properties can be imparted to the frame 220 and the resin sheet 230. For example, by increasing the rigidity of the frame 220 and increasing the oxygen permeability of the resin sheet 230, a culture vessel 100 with both rigidity and oxygen permeability can be obtained.

[0022] The 4-methyl-1-pentene (co)polymer that forms the material for the resin sheet 230 can be a homopolymer of 4-methyl-1-pentene, or a copolymer of 4-methyl-1-pentene with ethylene and an α-olefin (excluding 4-methyl-1-pentene) having 3 to 20 carbon atoms.

[0023] The above α-olefins may be linear, branched, or cyclic olefins. Examples of linear α-olefins include propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, and 1-eicosene. Examples of branched α-olefins include 3-methyl-1-butene, 3-methyl-1-pentene, 3-ethyl-1-pentene, 4,4-dimethyl-1-pentene, 4-methyl-1-hexene, 4,4-dimethyl-1-hexene, 4-ethyl-1-hexene, and 3-ethyl-1-hexene. Examples of cyclic olefins, specifically α-olefins, include cyclopentene, cyclohexene, cycloheptene, norbornene, 5-methyl-2-norbornene, tetracyclododecene, and vinylcyclohexane. Of these, homopolymers of 4-methyl-1-pentene, copolymers of 4-methyl-1-pentene and ethylene, and copolymers of 4-methyl-1-pentene and propylene are preferred, copolymers of 4-methyl-1-pentene and ethylene, and copolymers of 4-methyl-1-pentene and propylene are more preferred, and copolymers of 4-methyl-1-pentene and propylene are even more preferred.

[0024] From the viewpoint of creating aerobic conditions inside the containment section 210 to enhance the metabolic activity of microorganisms, the resin sheet 230 has an oxygen permeability of 150 cm². 3 / (m 2 ×24hr×atm) or more than 90000cm 3 / (m 2 It is preferable that the value be less than or equal to 24 hours and atm, and 150 cm 3 / (m 2 ×24hr×atm) or more 54500cm 3 / (m 2 It is more preferable that it be less than or equal to 6000 cm² (×24hr × atm), and 6000 cm². 3 / (m 2 ×24hr×atm) or more 54500cm 3 / (m 2It is even more preferable that the oxygen permeability is less than or equal to (x24hr x atm). From the viewpoint of effectively increasing oxygen permeability, it is preferable that the resin sheet 230 is a sheet-like member containing a polymer having a structural unit derived from 4-methyl-1-pentene.

[0025] The oxygen permeability values ​​mentioned above shall be those measured using the differential pressure gas permeability measurement method at a temperature of 37°C and relative humidity of 0%. The equipment used for measurement is not particularly limited as long as it employs the differential pressure gas permeability measurement method; for example, the BT-3MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd. can be used. The measurement sample shall be prepared by cutting a 90 x 90 mm test piece from resin sheet 230, with a measurement area diameter of 70 mm (permeable area of ​​38.46 cm²). 2 ) When high oxygen permeability is expected, an aluminum mask should be applied to the measurement sample beforehand, and the actual permeable area should be 5.0 cm². 2 It is preferable to do so.

[0026] The thickness of the resin sheet 230 is not particularly limited, but is preferably 10 μm to 500 μm, more preferably 20 μm to 300 μm, and even more preferably 20 μm to 200 μm. The greater the thickness, the stronger the resin sheet 230 can be. The less the thickness, the greater the oxygen permeability of the resin sheet 230 can be.

[0027] The adhesive layer 240 adheres the resin sheet 230 to the underside of the bottom plate portion of the housing 210. Known adhesives such as acrylic, silicone, urethane, and rubber-based adhesives can be used for the adhesive layer 240. Of these, acrylic and silicone-based adhesives are preferred.

[0028] Furthermore, the substrate 200 may have a coating layer 300 made of a hydrophilic material on the inner surface of the containment section 210 to enhance the adhesion of microorganisms.

[0029] The size of each containment section 210 is not particularly limited, but the area of ​​the resin sheet 230 (oxygen permeable layer) that constitutes the bottom surface of each containment section 210 when the culture vessel 100 is viewed from above is 8 mm². 2 960mm or more 2 The following is preferable: 30 mm 2 960mm or more 2 More preferably, the following: 191 mm 2 960mm or more 2 The following is even more preferable: The smaller the area of ​​the bottom surface of the containment section 210, the easier it is to culture microorganisms on a microscale. Cultivating microorganisms on a microscale has the advantage of making it easier to control the culture conditions and allowing reactions and analyses to be performed quickly and with high accuracy. By making the area of ​​the bottom surface of the containment section 210 appropriately large, oxygen can be efficiently taken in from the resin sheet 230, and the efficiency of microbial cultivation can be increased.

[0030] Furthermore, the maximum depth of the gap between the edge of the opening of each containment section 210 and the resin sheet 230 is preferably 4.1 mm or more and 16.7 mm or less, more preferably 8.3 mm or more and 16.7 mm or less, and even more preferably 12.5 mm or more and 16.7 mm or less. The deeper the containment section 210, the larger the amount of culture medium (culture solution) that can be contained in the containment section 210, and the larger the amount of nutrients supplied from the culture medium to the microorganisms, thereby increasing the efficiency of microbial cultivation. The shallower the containment section 210, the easier it becomes to cultivate microorganisms on a microscale.

[0031] Furthermore, the area [mm] of the resin sheet 230 (oxygen permeable layer) constituting the bottom member of the culture container 100 when viewed from above, relative to the depth [mm] of each storage section 210. 2The ratio of the resin sheet 230 (oxygen permeable layer) constituting the bottom member is preferably 0.40 mm or more and 230 mm or less, more preferably 1.70 mm or more and 230 mm or less, and even more preferably 1.70 mm or more and 80 mm or less. The larger the above ratio of the resin sheet 230 (oxygen permeable layer) constituting the bottom member, the more efficiently oxygen can be taken into the interior of the containment section 210, so that even if the depth of the containment section 210 is increased and the volume is increased, sufficient oxygen can be supplied to the cultured microorganisms. Therefore, by increasing the depth of the containment section 210 (increasing the above ratio), it is possible to increase the amount of culture medium (culture solution) contained in the containment section 210 while also supplying sufficient oxygen to the microorganisms, thereby significantly increasing the culture efficiency of the microorganisms.

[0032] The volume of each storage compartment 210 is 130 mm 3 The above is 16100mm 3 Preferably, it is 500 mm 3 The above is 16100mm 3 The following is more preferable: 3100mm 3 The above is 16100mm 3 The following is even more preferable: The larger the volume of the containment section 210, the larger the amount of culture medium (culture solution) that can be contained in the containment section 210, and the larger the amount of nutrients supplied from the culture medium to the microorganisms, thereby increasing the efficiency of microbial cultivation. The smaller the volume of the containment section 210, the easier it becomes to cultivate microorganisms on a microscale.

[0033] Also, the volume of each culture vessel 100 [mm²] 3 The area of ​​the resin sheet 230 (oxygen permeable layer) constituting the bottom member when the culture vessel 100 is viewed from above [mm²] 2 The ratio of ] is 0.01 mm -1 More than 0.25mm -1 Preferably, the following: 0.02 mm -1 0.15mm or more -1 The following is more preferable: 0.05 mm -1 The above is 0.09mm -1The following is even more preferable: The larger the proportion of the resin sheet 230 (oxygen permeable layer) that constitutes the bottom member, the more efficiently oxygen can be taken into the interior of the containment section 210, sufficient oxygen can be supplied to the microorganisms, and the efficiency of microbial cultivation can be significantly increased.

[0034] 2. Method for static culture of microorganisms and method for producing proteins In this embodiment, microorganisms are cultured statically using the culture vessel described above.

[0035] Microbial culture can be carried out by placing the culture medium (culture solution) and the target microorganism in each of the containment compartments 210 and placing them in the culture space of a culture device (such as a constant temperature room) set to a predetermined temperature and other conditions. At this time, the microorganisms are cultured statically without applying artificial shaking using a microplate shaker or the like. In this embodiment, culture is carried out by placing the culture medium and the target microorganism in multiple containment compartments 210. This enables efficient static culture of microorganisms.

[0036] The type of microorganism is not limited, and a wide range of multicellular organisms such as bacteria, archaea, fungi, protists, viruses, and microalgae can be used. Examples of bacteria include Escherichia coli, Bacillus subtilis, and Corynebacterium. Examples of archaea include methanogenic bacteria. Examples of fungi include yeast and mold. Examples of protists include amoebas, Paramecium, and malaria parasites. Examples of viruses include influenza viruses, human immunodeficiency virus (HIV), and coronaviruses. Examples of microalgae include Chlorella, cyanobacteria, and diatoms. Of these, bacteria and fungi are preferred, Escherichia coli, Bacillus subtilis, Corynebacterium, and yeast are more preferred, and Escherichia coli is even more preferred. The static culture method for microorganisms according to this embodiment can be applied to a wide variety of microorganisms, including microorganisms different from the cellulose-producing microorganisms described in Patent Document 5.

[0037] The size of the microorganisms is not particularly limited, but is preferably between 20 nm and 10 μm, more preferably between 300 nm and 6.4 μm, and even more preferably between 1000 nm and 5 μm.

[0038] The microorganisms are preferably aerobic microorganisms or microorganisms that can be cultured under aerobic conditions.

[0039] The maximum value of the gap between the top surface of the culture medium in each containment section 210 and the resin sheet 230, which is the liquid height of the culture medium (culture solution), is preferably 4.1 mm or more and 16.7 mm or less, more preferably 8.3 mm or more and 16.7 mm or less, and even more preferably 12.5 mm or more and 16.7 mm or less. The larger the amount of culture medium, the greater the amount of nutrients supplied from the culture medium to the microorganisms, thereby increasing the efficiency of microbial cultivation.

[0040] Furthermore, the area [mm²] of the resin sheet 230 (oxygen permeable layer) that constitutes the bottom member of the culture vessel 100 when viewed from above, relative to the liquid height [mm²] of each culture medium. 2 The ratio of the resin sheet 230 (oxygen permeable layer) constituting the bottom member is preferably 0.40 mm or more and 230 mm or less, more preferably 1.70 mm or more and 230 mm or less, and even more preferably 1.70 mm or more and 80 mm or less. The larger the above ratio of the resin sheet 230 (oxygen permeable layer) constituting the bottom member, the more efficiently oxygen can be taken into the interior of the containment section 210, so that even if the depth of the containment section 210 is increased and the volume is increased, sufficient oxygen can be supplied to the cultured microorganisms. Therefore, by increasing the depth of the containment section 210 (increasing the above ratio), it is possible to increase the amount of culture medium (culture solution) contained in the containment section 210 while also supplying sufficient oxygen to the microorganisms, thereby significantly increasing the culture efficiency of the microorganisms.

[0041] The amount of culture medium (culture solution) to be contained in each of the storage compartments 210 is 30 mm 3 The above is 16100mm 3 Preferably, it is 120 mm 3 The above is 16100mm 3The following is more preferable: 370 mm 3 The above is 16100mm 3 The following is even more preferable: The larger the amount of culture medium, the greater the amount of nutrients supplied to the microorganisms from the medium, thereby increasing the efficiency of microbial cultivation.

[0042] Also, the volume of each culture medium (culture solution) [mm²] 3 The area of ​​the resin sheet 230 (oxygen permeable layer) that constitutes the bottom member when the culture vessel 100 is viewed from above [mm²] 2 The ratio of ] is 0.05 mm -1 0.24mm -1 Preferably, the following: 0.05 mm -1 The above is 0.12mm -1 The following is more preferable: 0.05 mm -1 0.08mm or more -1 The following is even more preferable: The larger the proportion of the resin sheet 230 (oxygen permeable layer) constituting the bottom member, the more efficiently oxygen can be taken into the interior of the containment section 210, sufficient oxygen can be supplied to the microorganisms, and the efficiency of microbial cultivation can be significantly increased.

[0043] The concentration of microorganisms administered (seeded) into each containment section 210 is preferably such that the optical density (OD) is 0.01 or more and 1 or less, and more preferably such that the optical density (OD) is 0.05 or more and 0.2 or less.

[0044] The culture conditions are not particularly limited; the temperature and culture time should be determined according to the type of microorganism being cultured.

[0045] After culturing, the contents of the containment section 210 may be processed. Examples of processing include the recovery and measurement of the cultured microorganisms, the products produced by the microorganisms, and the culture medium containing the microorganisms. The recovered microorganisms may be stored or transported, or further processing may be performed on the recovered microorganisms. The above measurements may be performed on the recovered microorganisms or culture medium, or the inside of the containment section 210 may be measured directly. Examples of the above measurements include the measurement of the amount of microorganisms and the amount of protein expression by optical, physical, or chemical methods.

[0046] Alternatively, a protein may be synthesized (expressed) by microorganisms cultured in this manner, and the protein may be recovered from the recovered microorganisms. In this case, the microorganisms used may be those that have been transformed by introducing an expression vector containing a gene and its promoter for expressing the target protein.

[0047] In this embodiment, since the culture section uses a culture vessel 100 having an oxygen permeable layer, microorganisms can be effectively cultured even by static culture without shaking. Furthermore, since culture is performed simultaneously in multiple culture sections (container sections 210), the culture efficiency of microorganisms can be increased. Because shaking is unnecessary, a dedicated device is not required, and contamination due to splashing of liquid during shaking does not occur. Moreover, since a shaking culture device is not required, culture vessels 100 can be arranged vertically or adjacent culture vessels 100 can be placed touching each other for culture, thereby significantly increasing the number of cultures per unit area.

[0048] In this embodiment, microorganisms can also be cultured on a microscale using microplates. Conventionally, it was thought that vigorous shaking was necessary for culturing using microplates. In contrast, the fact that static culture can be performed without shaking by providing an oxygen permeable layer, and that logarithmic growth of microorganisms is possible, is a remarkable discovery.

[0049] 3. Other Embodiments It should be noted that each of the embodiments described above is merely an example of the present invention, and the present invention is not limited to the embodiments described above. It goes without saying that many other diverse embodiments are possible within the scope of the concept of the present invention.

[0050] For example, in the above embodiment, the resin sheet 230 constituting the bottom surface of the containment section 210 is the oxygen permeable layer, but a resin sheet or nonwoven fabric covering the top surface of the containment section 210 may also be used as the oxygen permeable layer. Alternatively, the frame 220 may be formed from an oxygen permeable material, and the thinner parts of its bottom and sides may be used as oxygen permeable layers to supply oxygen to the inside of the containment section 210. However, microorganisms in culture tend to be localized at the bottom of the containment section 210, and especially if growth is poor, their migration decreases and they tend to sink to the bottom. From the viewpoint of efficiently supplying oxygen to microorganisms present at the bottom and improving culture efficiency, it is preferable to make the bottom surface of the containment section 210 the oxygen permeable layer.

[0051] Furthermore, it is preferable that the containment section 210 has a transparent section whose total light transmittance, as measured in accordance with JIS K 7361-1:1997, is 70% or more and 100% or less. The containment section 210 having a transparent section allows for optical measurement without recovering microorganisms or culture media. For example, the resin sheet 230 may be made of transparent resin, or the frame 220 may be made of transparent resin. [Examples]

[0052] The present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0053] The LB medium used in the following experiments was prepared by dissolving Difco LB Broth Miller (Luria-Bertani) ("Difco" is a registered trademark of Becton Dickinson) in deionized water to a concentration of 25 g / L, and then autoclaving it at 121°C for 20 min.

[0054] [Experiment 1] A 4-methyl-1-pentene polymer (weight-average molecular weight (Mw) of 428,000, molecular weight distribution (Mw / Mn) of 4.1) was fed into an extruder with a T-die and extruded at an extrusion temperature of 270°C. The temperature of the rolls that came into contact with the extruded material afterward was set to 60°C, and the rotation speed of the rolls was adjusted to obtain a 4MP1 film with a thickness of 50 μm.

[0055] The oxygen permeability of the obtained film was measured using a differential pressure gas permeability measuring device (Toyo Seiki Seisakusho, MT-C3) in accordance with the JIS K 7126-1 (differential pressure method) standard under conditions of 37°C and 0% humidity. The diameter of the measuring section was 70 mm (permeable area was 38.46 cm²). 2 ) was used. Since a large oxygen permeability coefficient was expected, an aluminum mask was applied to the sample beforehand, and the actual permeability area was set to 5.0 cm². 2 The measured oxygen permeability was 38,200 cm³. 3 / (m 2 It was ×24h×atm).

[0056] The oxygen permeability of commercially available polyethylene terephthalate (PET) film (manufactured by STEM Co., Ltd., P96P01S) was similarly determined at 40 cm². 3 / (m 2 It was ×24h×atm).

[0057] A 4MP1 film was cut to a size of 8cm x 12cm and attached to the bottom of a 384-well polystyrene container frame using medical adhesive (manufactured by 3M) to create a culture plate (culture vessel). The culture plates were then placed in gamma-ray resistant bags and sterilized by irradiation with 10kGy of gamma rays.

[0058] A single colony of Escherichia coli strain BL21(DE3) was inoculated into 5 mL of LB medium and incubated overnight at 37°C. Next, the culture solution was diluted with LB medium to an OD600 of 0.02, and 50 μL was seeded into each well of a sterile culture plate.

[0059] Subsequently, a PET film was tightly applied to the bottom surface of the culture plate, covering only some of the wells and not the others. Similarly, a PET film was tightly applied to the top surface of the culture plate, covering only some of the wells and not the others. In this way, the wells shown in Table 1 (multiple wells in each case) were prepared.

[0060] [Table 1]

[0061] Next, the culture plates were incubated statically at 30°C in a plate reader (MOLECULAR DEVICES, SpectraMax i3x). The absorbance (OD600) was measured every 10 minutes after irradiation with light at a wavelength of 600 nm. The average absorbance measured from each well was calculated for each culture condition. The results are shown in Figures 3A and 3B.

[0062] As shown in Figures 3A and 3B, under both conditions with and without top ventilation, absorbance was higher when a film with high oxygen permeability (oxygen permeable layer) was placed at the bottom (with bottom ventilation), indicating higher microbial culture efficiency. Furthermore, compared to the condition without top ventilation, absorbance was higher with top ventilation, indicating higher microbial culture efficiency.

[0063] [Experiment 2] We prepared a culture plate for E. coli before seeding, which was created in Experiment 1 by tightly sealing a 4MP1 sheet in a 384-well container (hereinafter simply referred to as the "4MP1 plate"), and a commercially available polystyrene (PS) culture plate (Corning, 384-well polystyrene plate #4515) (hereinafter simply referred to as the "PS plate"). The oxygen permeability of the bottom surface of the PS plate was determined according to the JIS K 7126-1 differential pressure method standard (temperature 37°C, humidity 0%RH, O2 supplied and permeated from the outside of the container), and it was found to be 200 cm³. 3 / (m 2 It was (x24h x ATM).

[0064] A single colony of Escherichia coli DH5α strain was inoculated into 5 mL of LB medium and incubated overnight at 37°C. Next, the culture solution was diluted with LB medium to an OD600 of 0.01. 20 μL of the diluted culture solution was then seeded into each well of each plate, which had been sterilized in the same manner as in Experiment 1.

[0065] Subsequently, a nonwoven fabric sheet (Nunc Sealing Tape, White Rayon Breathable Sterile, manufactured by Thermo SCIENTIC) was attached to the top surface to prevent evaporation of the liquid components, and the samples were incubated statically in a constant temperature bath (CN-40A, manufactured by Mitsubishi Chemical Engineering Corporation) at 37°C for 12 hours.

[0066] Next, the nonwoven fabric sheet on the top surface was peeled off, and the culture medium in the wells was pipetteed. The absorbance of light at a wavelength of 600 nm (OD600) was measured using a micro-spectrophotometer (Thermo SCIENTIC, NanoDrop One). Figure 4 shows the mean and standard deviation (SD) of the OD600 measured for each of the eight wells.

[0067] As shown in Figure 4, the 4MP1 plate, which had a higher oxygen permeability in the oxygen-permeable layer, showed higher microbial culture efficiency than the PS plate.

[0068] [Experiment 3] We prepared a 4MP1 plate prepared in Experiment 2 and a commercially available culture plate (SARSTEDT, LUMOX multiwell, Cell culture plate, with foil base, 384 wells) (hereinafter simply referred to as "LUMOX plate"). The oxygen permeability at the bottom of the LUMOX plate was determined using the same method as in Experiment 2, and it was found to be 5950 cm². 3 / (m 2 It was ×24h×atm).

[0069] A single colony of Escherichia coli strain BL21(DE3) was inoculated into 5 mL of LB medium and incubated overnight at 37°C. Next, the culture solution was diluted with LB medium to an OD600 of 0.01. 50 μL of the diluted culture solution was then seeded into each well of each plate, which had been sterilized in the same manner as in Experiment 1.

[0070] Subsequently, a nonwoven fabric sheet (Nunc Sealing Tape, White Rayon Breathable Sterile, manufactured by Thermo SCIENTIC) was attached to the top surface to prevent evaporation of the liquid components, and the samples were incubated statically in a constant temperature bath (CN-40A, manufactured by Mitsubishi Chemical Engineering Corporation) at 37°C for 26 hours.

[0071] At the start of culture, and at 4, 8, and 26 hours after the start of culture, the nonwoven fabric sheet on top was removed, the culture medium in the wells was pipetteed, and the absorbance of light at a wavelength of 600 nm (OD600) was measured using a micro-spectrophotometer (Thermo SCIENTIC, NanoDrop One). Figure 5 shows the average OD600 values ​​measured for eight wells at each time point.

[0072] As shown in Figure 5, 4MP1 plates with higher oxygen permeability in the oxygen permeable layer showed higher microbial culture efficiency than LUMOX plates.

[0073] [Experiment 4] The 4MP1 film prepared in Experiment 1 was cut to a size of 8 cm x 12 cm and attached to the bottom of polystyrene 6-well, 24-well, 96-well, and 384-well container frames using medical adhesive (3M Corporation) to create culture plates (culture vessels) (hereinafter, these plates will simply be referred to as "6-well plate," "24-well plate," "96-well plate," and "384-well plate").

[0074] A single colony of Escherichia coli BL21(DE3) strain was inoculated into 5 mL of LB medium and incubated overnight at 37°C. Next, the culture solution was diluted with LB medium to an OD600 of 0.01. The diluted culture solution was then seeded into each well of each plate, which had been sterilized in the same manner as in Experiment 1, so that the liquid height from the bottom surface was 0.8 cm. Table 2 shows the size of the wells on each plate (bottom surface area, depth of the culture area, volume of the culture area) and the amount of culture solution (liquid height, liquid volume).

[0075] [Table 2]

[0076] Subsequently, a nonwoven fabric sheet (Nunc Sealing Tape, White Rayon Breathable Sterile, manufactured by Thermo SCIENTIC) was attached to the top surface to prevent evaporation of the liquid components, and static incubation was performed at 37°C in a constant temperature bath (CN-40A, manufactured by Mitsubishi Chemical Engineering Corporation).

[0077] At the start of culture, and at 2, 4, 6, and 21 hours after the start of culture, the nonwoven fabric sheet on top was removed, the culture medium in the well was pipetteed, and the absorbance of light at a wavelength of 600 nm (OD600) was measured using a micro-spectrophotometer (Thermo SCIENTIC, NanoDrop One). Figure 6 shows the average values ​​of the OD600 measured at each time point.

[0078] As shown in Figure 6, 4MP1 plates with a larger area of ​​the oxygen permeable layer at the bottom relative to the depth of the containment section and the liquid height of the culture medium showed higher microbial culture efficiency than 4MP1 plates with a smaller area of ​​the oxygen permeable layer.

[0079] [Experiment 5] The 4MP1-384 plates prepared in Experiment 4 were then placed in gamma-ray resistant bags and sterilized by irradiating with 10 kGy of gamma rays.

[0080] A single colony of Escherichia coli BL21(DE3) strain was inoculated into 5 mL of LB medium and incubated overnight at 37°C. Next, the culture solution was diluted with LB medium to an OD600 of 0.01. The diluted culture solution was then seeded into each well of the plate, which had been sterilized in the same manner as in Experiment 1, to 1 / 2, 3 / 4, and the full volume of the well (hereinafter, these wells will simply be referred to as "1 / 2 volume well," "3 / 4 volume well," and "full volume well"). Table 3 shows the size of the wells on the plate (bottom area, depth of the culture area, volume of the culture area) and the volume of the culture solution (liquid height, liquid volume).

[0081] [Table 3]

[0082] Subsequently, a nonwoven fabric sheet (Nunc Sealing Tape, White Rayon Breathable Sterile, manufactured by Thermo SCIENTIC) was attached to the top surface to prevent evaporation of the liquid components, and static incubation was performed at 37°C in a constant temperature bath (CN-40A, manufactured by Mitsubishi Chemical Engineering Corporation).

[0083] At the start of culture, and at 2, 4, and 21 hours after the start of culture, the nonwoven fabric sheet on top was removed, the culture medium in the well was pipetteed, and the absorbance of light at a wavelength of 600 nm (OD600) was measured using a micro-spectrophotometer (Thermo SCIENTIC, NanoDrop One). Figure 7 shows the average values ​​of the OD600 measured at each time point.

[0084] As shown in Figure 7, optimizing the amount of culture medium improved the efficiency of microbial cultivation.

[0085] [Experiment 6] L-amino acid α-ligase-expressing Escherichia coli was inoculated into LB medium to achieve an initial OD600 of 0.1 and cultured in a baffled flask at 37°C for 2.5 hours. Subsequently, isopropyl-β-thiogalactopyranoside (IPTG) was added to induce L-amino acid α-ligase expression to a final concentration of 0.1 mM.

[0086] Subsequently, a portion of the culture medium was dispensed into 4MP1-384 plates prepared in Experiment 4, with 50 μL / well portions. The fraction remaining in the flask was cultured at 25°C for 22 hours with shaking at 120 rpm, while the 4MP1-384 plates were cultured statically at 25°C for 22 hours.

[0087] After 22 hours of incubation, 1 mL of culture medium was taken from both the flask and the culture plate, placed in an Eppendorf tube, centrifuged, and then resuspended in 1 mL of pure water. The suspension was sonicated, and the soluble and insoluble fractions were extracted by centrifugation. The extracted fractions were mixed with a reducing agent (ThermoFisher, NuPAGE Sample Reducing Agent (10x)) and a sample buffer (TCI, 4x SDS-PAGE Sample buffer), and heat-treated at 75°C for 10 minutes. Protein analysis was then performed by SDS-PAGE.

[0088] Figure 8 shows the SDS-PAGE results for each fraction. M is the molecular weight marker. A band corresponding to the molecular weight of L-amino acid α-ligase (approximately 55 kDa) was observed with similar intensity in both the soluble fraction from the flask cultured with shaking and the soluble fraction from the 4MP1-384 plate cultured under static conditions (the framed area in Figure 8). This result indicates that protein expression levels are similar in both flask culture and static culture on the 4MP1-384 plate. [Industrial applicability]

[0089] The culture vessel according to the present invention can be applied to the static culture of various microorganisms. [Explanation of symbols]

[0090] 100 culture vessels 200 Base material 210 Storage Unit 220 Frame 222 Frame section 224 wells 230 resin sheets 240 Adhesive layer 300 coating layers

Claims

1. A culture vessel for static culture of microorganisms, Each has multiple culture sections, each with an oxygen-permeable layer. All of the aforementioned oxygen permeable layers measured at 37°C and 0% humidity, with an oxygen permeability of 150 cm³. ３ / (m ２ ・24h・ATM) or more 90000cm ３ / (m ２ - 24 hours (atm) or less Culture container.

2. The oxygen permeable layer contains a 4-methyl-1-pentene polymer. The culture vessel according to claim 1.

3. The 4-methyl-1-pentene polymer is at least one polymer selected from the group consisting of a homopolymer of 4-methyl-1-pentene and copolymers of 4-methyl-1-pentene with ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). The culture vessel according to claim 2.

4. Having five or more of the culture sections, The culture vessel according to claim 1.

5. The culture section has the oxygen permeable layer as a bottom member. The culture vessel according to claim 1.

6. The culture section has an area of ​​8 mm² of the oxygen permeable layer that constitutes the bottom member when the culture container is viewed from above. ２ 960mm or more ２ The following is: The culture vessel according to claim 5.

7. The culture section is defined as the area of ​​the oxygen permeable layer constituting the bottom member when the culture container is viewed from above, relative to the depth of the culture section [mm]. ２ The ratio of ] is 0.40 mm or more and 230 mm or less. The culture vessel according to claim 5.

8. The culture section has a ratio of the area [mm ３ of the oxygen permeable layer constituting the bottom member when the culture container is viewed in plan view to the volume [mm ２ of the culture section, which is 0.01 mm －１ or more and 0.25 mm －１ or less, The culture vessel according to claim 5.

9. The microorganism is at least one selected from the group consisting of bacteria, archaea, fungi, protists, viruses, and microalgae. The culture vessel according to claim 1.

10. A step of preparing a culture vessel according to any one of claims 1 to 9, The process includes the step of statically culturing microorganisms inside the culture section of the culture vessel, A method for static culture of microorganisms.

11. The culture section has the oxygen permeable layer as a bottom member, In the static culture step, the area of ​​the oxygen permeable layer having as the bottom member [mm] is relative to the liquid height [mm] of the culture medium containing the microorganisms. ２ The ratio of ] is 0.40 mm or more and 230 mm or less. A method for static culture of microorganisms according to claim 10.

12. The culture section has the oxygen permeable layer as a bottom member, In the static culture step, the volume of the culture medium containing the microorganisms [mm²] ３ The area of ​​the oxygen permeable layer having as the bottom member [mm²] ２ The ratio of ] is 0.05 mm －１ The above is 0.24 mm. －１ The following is: A method for static culture of microorganisms according to claim 10.

13. The process includes a step of treating the contents of the culture section after the step of static culture. A method for static culture of microorganisms according to claim 10.

14. A step of preparing a culture vessel according to any one of claims 1 to 9, The process includes the step of statically culturing microorganisms inside the culture section of the culture vessel, A method for producing proteins.

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