CN111235014A - Culture dish and method for rapidly testing bacteriostatic ability of bacteriostatic substance by using culture dish - Google Patents
Culture dish and method for rapidly testing bacteriostatic ability of bacteriostatic substance by using culture dish Download PDFInfo
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- CN111235014A CN111235014A CN202010181639.XA CN202010181639A CN111235014A CN 111235014 A CN111235014 A CN 111235014A CN 202010181639 A CN202010181639 A CN 202010181639A CN 111235014 A CN111235014 A CN 111235014A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Abstract
The invention belongs to the field of microorganisms, and particularly relates to a culture dish and a method for rapidly testing bacteriostatic ability of a bacteriostatic substance by using the culture dish. The specific technical scheme is as follows: a culture dish comprises a dish plate, wherein the periphery of the dish plate extends upwards to form a side wall to form a space for containing contents, and the space is divided into a plurality of culture sections which are not communicated with each other by partition plates; the height of the partition is 1/2-4/5 of the height of the side wall of the dish plate; the culture dish also comprises a dish cover, and the height of the dish cover is 1.3-2 times of the height of the side wall of the dish plate; when the ware lid covers the ware board, with ware board lateral wall clearance fit, the ware lid is kept away from ware board one side bottom surface and is run through the opening that sets up the area sealed lid, opening and hollow pipette clearance fit, and the pipette can shift the content of ware board between different intervals. The invention provides a brand-new culture dish, which can realize that a plurality of groups of contrast tests are carried out in the same culture dish under the same test condition; and the convenient operation without uncovering can be realized, and the pollution possibility in the operation is greatly reduced.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a culture dish and a method for rapidly testing bacteriostatic ability of a bacteriostatic substance by using the culture dish.
Background
The cell culture dish is a common vessel in a laboratory and is generally composed of a dish plate and a dish cover. In the specific operation, the inoculum is inoculated on the dish plate, and then the dish cover is covered for culture. The prior culture dish has the following defects: the single culture medium consumption is large; when different inocula are inoculated in different areas of a single culture dish to form a control, information exchange between the inocula can occur, and the possibility of contamination exists. In addition, after the inoculation is completed, if a subsequent operation is required, the conventional culture dish can be carried out only by opening the dish cover, and although the step is carried out in a sterile environment such as a sterile table, the step still has a high possibility of contamination. Thus, providing a culture dish that overcomes the above-mentioned drawbacks would greatly facilitate cell/bacteria experiments.
Disclosure of Invention
The invention aims to provide a culture dish and a method for rapidly testing bacteriostatic ability of a bacteriostatic substance by using the culture dish.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a culture dish comprises a culture dish plate made of transparent materials, wherein the periphery of the culture dish plate extends upwards to form a side wall to form a space for containing contents, and the space is divided into a plurality of culture sections which are not communicated with each other by partition plates; the height of the partition is 1/2-4/5 of the height of the side wall of the dish plate;
the culture dish comprises a dish cover which is made of transparent materials and can cover the dish plate, and the height of the dish cover is 1.3-2 times of the height of the side wall of the dish plate; the ware lid is bald precious lid shape, inside cavity shape and ware board adaptation, when the ware lid covers the ware board, with ware board lateral wall clearance fit, the ware lid is kept away from ware board one side bottom surface and is run through the opening that sets up the sealed lid of area, opening and hollow pipette clearance fit, the pipette can shift the content of ware board between different intervals.
Preferably, the partition plates are symmetrically arranged inside the dish plate, and the partition plates are converged at the center of the dish plate to form the isolation column.
Preferably, when the dish cover covers the dish plate, the opening arranged on the dish cover is positioned right above the isolation column.
Preferably, the pipette comprises a penetrating part penetrating through the opening of the dish cover, the penetrating part is made of elastic materials, and a suction part which can be directly contacted with the content of the dish plate is fixedly connected below the penetrating part;
when the pipette is inserted into the opening, the central axis of the penetrating part is parallel to the side wall of the dish plate, and the suction part is inclined relative to the side wall of the dish plate at an angle which can ensure that the suction part sucks the contents in each section of the dish plate.
Preferably, a limiting block is fixedly connected to the outer tube wall of the penetrating part of the pipette, and the sum of the widths of the limiting block and the corresponding part of the penetrating part is greater than the diameter of the opening on the dish cover; the dish cover is correspondingly provided with a limiting groove through which the limiting block can pass, and the limiting groove is sealed by a sealing cover which can be opened and closed.
Preferably, the suspension end of the pipette penetrating part is detachably connected with a rotating button, and the diameter of the rotating button is larger than the opening on the dish cover.
Preferably, the distance between the rotating button and the limiting block is larger than the maximum distance between the suspension end of the suction part and the surface of the content in the dish plate.
Preferably, a liquid inlet with a sealing cover is arranged between the rotating button and the limiting block and on the side wall of the penetrating part in a penetrating mode.
Correspondingly, the method for testing the bacteriostatic ability of the bacteriostatic protein by using the culture dish comprises the following steps:
(1) transferring the gene of the bacteriostatic protein into bacteria to be inhibited;
(2) adding culture media suitable for the growth of the bacteria in the step (1) into each interval of the culture dish plate respectively, wherein the culture media at least comprise 1 liquid culture medium and 1 solid culture medium;
(3) inoculating the bacteria in the step (1) into the interval filled with the liquid culture medium of the dish plate, and performing shake culture;
(4) adding a bacteriostatic protein induction expression agent into a liquid culture medium containing bacteria by using a pipette, and performing shake culture;
(5) sucking the contents in the liquid culture medium in the step (4) by using a pipette, respectively inoculating the contents to the solid culture medium in other intervals of the dish plate, and continuously culturing;
(6) and observing the growth of bacteria on the solid culture medium in each interval.
Preferably, step (4) is performed during the log phase of bacterial growth.
The invention has the following beneficial effects:
1. the invention provides a brand-new culture dish, and multiple groups of contrast tests can be performed in the same culture dish under the same test conditions through the partition design of the culture dish. Through the matching design of the pipette and the dish cover, the convenient operation of uncovering is realized, and the pollution possibility in the operation is greatly reduced.
2. The culture dish and the method provided by the invention are adopted to verify the bacteriostatic effect of the bacteriostatic protein, so that the raw materials are greatly saved: the whole verification test can be completed by using the raw material of the previous culture dish. The operation is convenient, and the pipette can be operated by rotating; the pollution risk is low, and the culture dish does not need to be frequently uncovered for operation in the test process. Meanwhile, the invention greatly shortens the test time, and the general idea of verifying whether the substance has the bacteriostatic activity is as follows: transferring the gene of the substance into a certain bacterium, inducing and expressing the substance, separating and purifying the substance, and then independently using the substance to perform a bacteriostatic test. The invention combines expression and verification, thus saving test time and operation links. The invention can also save the test space and ensure the uniformity of the test conditions as much as possible. In addition, if the bacteriostatic protein has bacteriostatic activity, bacteria die or are in a low-activity state in the test process, and the subsequent harmless treatment difficulty is greatly reduced.
Drawings
FIG. 1 is a schematic view of the structure of a culture dish according to the present invention;
FIG. 2 is a view A-A of FIG. 1;
FIG. 3 is a schematic diagram showing the growth of E.coli containing pET32a plasmid;
FIG. 4 is a schematic diagram showing the growth of E.coli containing the recombinant plasmid pET32a-BNBD 4.
Detailed Description
As shown in fig. 1 and 2, a culture dish comprises a culture dish plate 10 made of transparent materials. The dish plate 10 is typically transparent glass or plastic, low cost, and easy to clean, sterilize, and observe. The dish plate 10 extends upward around the periphery to form a side wall, which constitutes a space for accommodating the contents. The space is divided into a plurality of culture areas which are not communicated with each other by the partition plates 11; the height of the partition plate 11 is 1/2-4/5 of the height of the side wall of the dish plate 10. The preferable scheme is as follows: the partition plates 11 are symmetrically arranged inside the dish plate 10, and the partition plates 11 are converged at the center of the dish plate 10 to form the isolation column 12. The dish plate 10 is in the shape of a bowl with a flat bottom and an upward opening. Referring to fig. 2, for example, 6 partitions 11 are provided in a well plate 10, and 6 partitions 11 and an isolation column 12 located at the center of the well plate 10 are provided in the well plate 10. Under the action of the partition board 11 and the isolation column 12, the interior of the dish plate 10 is divided into 6 sections which are independent and not communicated with each other. When filling the dish plate 10 with contents, care should be taken not to fill the contents too full. Particularly, when liquid contents are contained, in order to avoid that the liquid oscillates and crosses the partition plate 11 to enter other intervals when the shaking table culture or the culture dish moves, the containing amount of the liquid contents cannot exceed 4/5 of the volume of each interval, and the preferred containing volume is 1/2-2/3 of the volume of the corresponding interval.
The culture dish further comprises a dish cover 20 which is made of transparent materials and can cover the dish plate 10, and the material of the dish cover 20 is consistent with that of the dish plate 10. The height of the dish cover 20 is 1.3-2 times of the height of the side wall of the dish plate 10. The dish cover 20 is integrally in a bald cover shape, the shape of the inner cavity of the dish cover is matched with that of the dish plate 10, and the dish cover 20 is in clearance fit with the side wall of the dish plate 10 when covering the dish plate 10. It should be understood that, as shown in figure 1, the capsule 20 may just cover the dish plate 10 in terms of width; in terms of height, after the capsule 20 covers the capsule plate 10, a certain operation space is left between the capsule plate 10 and the capsule 20, and the space is provided by the height difference between the capsule plate 10 and the side wall of the capsule 20.
The bottom surface of the dish cover 20 on the side away from the dish plate 10 is provided with an opening with a sealing cover (the sealing cover is not shown in the figure) for the pipette 30 to enter and exit. The opening is a clearance fit with a hollow pipette 30, which pipette 30 can transfer the contents of the dish plate 10 between different compartments. The preferable scheme is as follows: when the partition plates 11 are symmetrically arranged, the opening formed in the dish cover 20 is just above the isolation column 12 after the dish cover 20 covers the dish plate 10.
When the mutual movement of the contents in each interval in the culture dish is not needed, the opening on the dish cover 20 can be directly sealed by the sealing cover, the culture can be carried out under appropriate conditions, and the using method is the same as that of a common culture dish. When the movement of the content is required, the sealing cover is opened, and the pipette 30 is inserted into the operation space reserved between the capsule 20 and the capsule plate 10 from the opening. The pipette 30 can rotate and move up and down within the opening because the pipette 30 is in a clearance fit with the opening. The operator controls the pipette 30 to rotate to the position above the content to be moved, so as to take the liquid, and then rotates the pipette 30 to the section where inoculation is required.
If the pipette 30 is configured to be straight, although the pipette 30 is generally made of flexible material (e.g., PVC) with certain flexibility, the working portion can be bent and inclined to some extent to perform the operations of liquid taking, liquid transferring, and inoculation. However, this presents operational difficulties and also increases the risk of bacterial contamination when the pipette 30 is tilted or deformed. Therefore, the more preferable scheme is: the pipette 30 is composed of a penetrating part 35 and a sucking part 34. When the portable household utensil is in operation, the penetrating part 35 penetrates through the opening of the utensil cover 20, and the penetrating part 35 is made of elastic materials (such as rubber); a suction part 34 capable of directly contacting with the content of the dish plate 10 is fixed below the penetrating part 35. It should be noted that the above description is merely for convenience of description, and does not mean that the pipette 30 is actually structurally divided into two parts, i.e., the penetrating part 35 and the suction part 34, which are independent of each other. In fact, in order to avoid leakage and facilitate work, the penetrating part 35 and the suction part 34 are integrally formed as a single body.
When the pipette 30 passes through the opening of the capsule 20, the central axis of the penetrating portion 35 is parallel to the side wall of the capsule plate 10, and the suction portion 34 is inclined with respect to the side wall of the capsule plate 10 at an angle that ensures that the suction portion 34 sucks the content in each section of the capsule plate 10. It should also be noted that the angle of inclination is not too large so as to increase the difficulty of removing the pipette 30 from the capsule 20; the preferred inclination angle is 30 ~ 45.
In this arrangement, the suction portion 34 can be easily rotated above the corresponding section of the dish plate 10 by rotating the portion of the through portion 35 located outside the dish lid 20; the penetrating part 35 is moved downwards until the sucking part 34 is contacted with the content in the interval, and then the penetrating part 35 is pressed or loosened, thus completing the sucking or releasing of the content.
The more preferable scheme is as follows: the outer wall of the penetrating part 35 of the pipette 30 is fixedly connected with a limiting block 33, and the sum of the widths of the corresponding parts of the limiting block 33 and the penetrating part 35 is larger than the diameter of the opening on the dish cover 20. The dish cover 20 is correspondingly provided with a limiting block 33 through a limiting groove (not shown), and the limiting groove is closed by a sealing cover which can be opened and closed. In this arrangement, the vessel cover 20 does not need to be uncovered during the whole culture and transfer process, and an opening for placing the pipette 30 on the vessel cover 20 does not need to be opened and closed. After the corresponding sections of the dish plate 10 are filled with the corresponding contents, the dish lid 20 is closed and the pipette 30 is mounted (or the dish lid 20 with the pipette 30 mounted thereon is closed), and then the test can be started. The pipette 30 is preferably mounted to the capsule 20 and then sterilized to avoid the risk of contamination associated with mounting the pipette 30 after sterilization.
At this time, the pipette 30 is fixed and limited on the capsule 20 under the action of the limiting block 33, and covers the opening on the capsule 20; the aspiration portion 34 of the pipette 30 is suspended and does not contact the contents of the well plate 10 in the respective compartments. When the content needs to be transferred, the sealing cover on the limiting groove is opened, the pipette 30 is rotated, the limiting block 33 penetrates through the limiting groove, and therefore the pipette 30 can do up-and-down reciprocating motion and autorotation motion relative to the dish cover 20 to achieve material transfer.
The more preferable scheme is as follows: for the convenience of operation, the suspended end (top end) of the penetrating part 35 of the pipette 30 is detachably connected with a rotating button 31, and the diameter of the rotating button 31 is larger than the opening on the capsule 20. When the operation is performed, the knob 31 is directly screwed. The pipette 30 is detachably connected to the knob 31, which also facilitates the replacement of the pipette 30. Specific detachable connection means are well established prior art, such as: the bottom of the rotating button 31 is provided with a hole for accommodating the pipette 30, the pipette 30 is in interference fit with the hole, and the penetrating part 35 of the pipette 30 is made of an elastic material and can be clamped into the hole in a squeezing mode and the like; the hole at the bottom of the rotary knob 31 may be provided with a screw, and the corresponding portion of the through portion 35 may be provided with a corresponding screw, and the through portion 35 may be directly screwed by rotation.
The distance between the rotating button 31 and the limiting block 33 is greater than the maximum distance from the suspension end of the suction part 34 to the surface of the content in the dish plate 10, so as to ensure that the suction part 34 can smoothly contact with the content.
The more preferable scheme is as follows: a liquid inlet 32 with a sealing cover is arranged between the rotating button 31 and the limiting block 33 and on the side wall of the penetrating part 35 in a penetrating way. When it is desired to add substances to the culture dish within the specified interval, this can be done via the inlet port 32.
The invention also provides a method for testing bacteriostatic ability of the bacteriostatic protein based on the culture dish, which specifically comprises the following steps:
(1) transferring the gene of the bacteriostatic protein into bacteria to be inhibited.
(2) And (2) respectively adding culture media suitable for the growth of the bacteria in the step (1) into each interval of the culture dish plate 10, wherein the culture media at least comprise 1 liquid culture medium and 1 solid culture medium, and the specific number is set according to the actual requirement.
(3) Respectively inoculating equal amount of the bacteria in the step (1) into the interval containing the liquid culture medium of the dish plate 10, and performing shake culture.
(4) Adding bacteriostatic protein induction expression agent into one interval filled with liquid culture medium, and continuously culturing by shaking table.
(5) The contents of the liquid medium are aspirated by using the pipette 30, and the liquid medium is inoculated to the solid medium in the other sections of the dish plate 10, respectively, and the culture is continued.
(6) And observing the growth of bacteria on the solid culture medium in each interval. If bacteria do not grow or grow obviously weaker than a control group (observing colony size and the like) on the solid culture medium corresponding to the group added with the bacteriostatic protein induced expression agent, the bacteriostatic protein has bacteriostatic ability on the bacteria. Then extracting the content of the corresponding liquid culture medium according to the actual test requirement, separating and purifying the bacteriostatic protein, and carrying out further other tests.
The method takes fig. 2(6 intervals) as an example, and specifically includes the following steps:
(1) transferring the gene of the bacteriostatic protein into bacteria to be inhibited, and culturing to obtain a seed solution of the recombinant strain.
(2) 6 sections, namely, a section a to f section, are provided in the dish plate 10. Wherein 2 intervals are filled with liquid culture medium (intervals a and b), 2 intervals are filled with solid culture medium (intervals c and d), 1 interval is used as a waste liquid area (interval e), and 1 interval is used as a liquid mixing area (interval f).
(3) Inoculating equal amount of seed liquid of the bacteria of step (1) into the interval (a, b) containing liquid culture medium of dish plate 10, and shake-culturing under appropriate conditions.
(4) Adding a proper amount of liquid culture medium to one of the intervals (interval b, control group) filled with the liquid culture medium by using the liquid inlet 32 on the pipette 30; then the pipette 30 is moved to the waste liquid zone (zone e), a small amount of pure water is added through the liquid inlet 32, the pipette 30 is cleaned, and the washing liquid is discharged into zone e; the pipette 30 is then transferred to another zone (zone a) containing liquid culture medium, and a proper amount of bacteriostatic protein is added through the liquid inlet 32 to induce the expression agent. The pipette 30 is then moved to the waste zone (zone e), a small amount of pure water is added through the inlet 32, the pipette 30 is washed and the wash solution is drained into zone e. And (5) continuing shaking culture.
Because the pipette 30 does not need to be contacted with the content (preferably, the contact is not needed, so that pollution is avoided), the pipette 30 can be suspended without opening the limit groove. By arranging the waste liquid area, on one hand, the closed operation is realized to the greatest extent, and on the other hand, toxic wastes possibly generated in the test are concentrated in the culture dish, so that the subsequent concentrated treatment is facilitated.
Because the bacteriostatic protein may generate a certain inhibiting effect or even a toxic effect on the growth of bacteria, the step (adding the bacteriostatic protein to induce the expression agent) is carried out when the strain grows to logarithmic phase and grows vigorously.
(5) After the culture for a certain period of time, the pipette 30 is used to aspirate the contents in the sections a and b, respectively, and the contents are inoculated into the sections c and d of the dish plate 10, respectively, and the culture is continued.
The contents of the interval a may be inoculated in both the intervals c and d of step (5). Prior to inoculation, the contents were diluted to different concentrations, with controls between each concentration. The specific dilution method comprises the following steps: the pipette 30 is moved to the section a, a certain amount of content is sucked, and then transferred to the section f (liquid mixing zone), an appropriate amount of diluent (such as liquid culture medium) is added through the liquid inlet 32, the liquid in the section f is repeatedly sucked up and released for several times through the pipette 30, and after the liquid is mixed uniformly, the liquid is transferred to the section c for inoculation. The same procedure was followed, but with another dilution of the strain in the d interval. Since the dilution liquid and the strain have the same composition but different concentration ratios, the dilution process can be performed in the interval f. For the convenience of operation, the preferred scheme is as follows: the side wall of the f interval is printed with scales (not shown in the figure). Because the invention is a test for preliminarily verifying the bacteriostatic effect, the dilution concentration does not need to be particularly accurate, and the concentrations are obviously different.
Of course, the operation of step (5) may also be: two or more intervals a are inoculated with the contents and two or more intervals b are inoculated with the contents, and the intervals c and d are controlled with each other. The method can be specifically set according to the test requirements.
(6) And observing the growth of bacteria on the solid culture medium in each interval.
The feasibility of the method of the invention is verified below with reference to specific examples.
Examples
1. Selecting healthy Hongyuan Maomai yaks of Sichuan Abao at 2.5 years old at random, collecting lung tissue samples by using a sterile tool after slaughter, removing bloodstain in DEPC water, and preserving in liquid nitrogen. Lung tissue was well ground using RNAiso Plus reagent and total RNA extracted from the tissue according to the total RNA extraction kit instructions. cDNA was synthesized according to reverse transcription kit instructions.
PCR primers were designed with reference to the sequence of the cattle BNBD4 gene (NM-174775.1) in Genebank, with the following primer information: TCTTCTCCAGCATCAGCC is used as a reference material; CTGGTTACGCCTCAGTCT is added.
And (3) carrying out PCR amplification reaction by using the wheat-hollow yak lung tissue cDNA as a template. The reaction system is 25 μ L, and the PCR reaction procedure is as follows: at 98 deg.C for 2 min; at 98 deg.C for 2 min; 55.6 ℃ for 10 s; at 72 ℃ for 10s, 35 cycles; 72 ℃ for 2 min; storing at 4 ℃. After the PCR amplification product is detected by 1% agarose gel electrophoresis, an amplification fragment of about 250bp is obtained.
2. The open reading frame is analyzed by using an ORF Finder program of NCBI, and the nucleotide sequence of the Maidong yak β -defensin 4 gene is translated into an amino acid sequence by using DNAMAN, wherein the open reading frame of β -defensin 4 gene is 195bp and can code 64 amino acids, and the amino acid sequence is MRLHHLLLALLFLVLSAGSGFTQVVRNPQSCRWNMGVCIPFLCRVGMRQIGTCFGPRVPCCRRW.
3. Inducible expression of the recombinant plasmid pET-32a-BNBD4 in E.coli BL21(DE 3).
And (3) optimizing a gene sequence according to an escherichia coli expression system, and constructing an expression vector. Carrying out double enzyme digestion on the pET-32a (+) vector plasmid and the target fragment by KpnI and EcoRI, connecting and transforming the plasmid into Escherichia coli TOP10, selecting positive clones, carrying out enzyme digestion verification, returning the recombinant plasmid after successful sequencing and identification. The whole gene synthesis is completed by the biotechnology limited company of the new industry of Beijing Ongkoku. The recombinant plasmid pET32a-BNBD4 was diluted to a plasmid solution of 4 ng/. mu.L using DEPC H2O. The transformed strain was obtained by chemical transformation of 100. mu.L of BL21(DE3) competent plasmid solution (2. mu.L). 200 μ L of the bacterial liquid was uniformly plated on LB plate (containing 50mg/ml Amp), and cultured at 37 ℃ for 12 hours. Then, single colony is selected and inoculated in LB liquid culture medium (containing 50mg/ml Amp), shaking culture is carried out for 6-8 h at 37 ℃ and 180r/min, and seed liquid is obtained.
4. To 50mL of LB liquid medium was added 1500. mu.L of the seed liquid described in step 3, and the mixture was cultured overnight at 37 ℃ and 180 r/min. Then 0.5mmol/L IPTG was added to induce expression for 6 h. Subsequently, the mixture was diluted by a factor of 10 to obtain 5 gradients (10) using LB liquid medium8CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL), 5. mu.L of each gradient was applied to LB plates (containing 100. mu.g/mL Amp and 0.5mmol/L IPTG), and cultured overnight at 37 ℃ to obtain a treated group. Coli containing pET32a empty plasmid was cultured under the same conditions as a control group. The results of the control group are shown in FIG. 3, and the results of the experimental group are shown in FIG. 4. As can be seen from FIGS. 3 and 4, the experimental group 107CFU/mL、106CFU/mL、105CFU/mL、104The colonies with the concentration of 4 CFU/mL are obviously less than that of a control group, the recombinant protein β -defensin 4 is proved to have obvious bacteriostatic action on escherichia coli, and the test method provided by the invention is proved to be quick, feasible and effective.
Claims (10)
1. A culture dish, characterized in that: the culture dish comprises a transparent culture dish plate (10), wherein the periphery of the dish plate (10) extends upwards to form a side wall to form a space for containing contents, and the space is divided into a plurality of culture sections which are not communicated with each other by partition plates (11); the height of the partition plate (11) is 1/2-4/5 of the height of the side wall of the dish plate (10);
the culture dish also comprises a transparent dish cover (20) capable of covering the dish plate (10), and the height of the dish cover (20) is 1.3-2 times of the height of the side wall of the dish plate (10); the dish lid (20) is inside cavity shape and dish board (10) adaptation, when dish lid (20) covers dish board (10), with dish board (10) lateral wall clearance fit, dish lid (20) are kept away from dish board (10) one side bottom surface and are run through the opening that sets up the sealed lid of area, opening and hollow pipette (30) clearance fit, pipette (30) can shift the content of dish board (10) between different intervals.
2. A culture dish according to claim 1, wherein: the partition plates (11) are symmetrically arranged inside the dish plate (10), the partition plates (11) are converged at the center of the dish plate (10) to form an isolation column (12), and the isolation column (12) is as high as the partition plates (11).
3. A culture dish according to claim 2, wherein: when the dish cover (20) covers the dish plate (10), the opening arranged on the dish cover (20) is positioned right above the isolation column (12).
4. A culture dish according to claim 3, wherein: the pipette (30) comprises a penetrating part (35) penetrating through an opening of the dish cover (20), the penetrating part (35) is made of elastic materials, and a suction part (34) which can be directly contacted with the content of the dish plate (10) is fixedly connected below the penetrating part (35);
when the pipette (30) passes through the opening, the central axis of the penetrating part (35) is parallel to the side wall of the dish plate (10), and the suction part (34) is inclined relative to the side wall of the dish plate (10) at an angle which can ensure that the suction part (34) sucks the contents in each section of the dish plate (10).
5. A culture dish according to claim 4, wherein: a limiting block (33) is fixedly connected to the outer tube wall of the penetrating part (35) of the pipette (30), and the sum of the widths of the corresponding parts of the limiting block (33) and the penetrating part (35) is larger than the diameter of an opening on the dish cover (20); the dish cover (20) is correspondingly provided with a limiting groove through which the limiting block (33) can pass, and the limiting groove is sealed by a sealing cover which can be opened and closed.
6. A culture dish according to claim 5, wherein: the suspension end of the penetrating part (35) of the pipette (30) is detachably connected with the rotating button (31), and the diameter of the rotating button (31) is larger than the opening aperture on the dish cover (20).
7. A culture dish according to claim 6, wherein: the distance between the rotating button (31) and the limiting block (33) is larger than the maximum distance from the suspension end of the suction part (34) to the surface of the content in the dish plate (10).
8. A culture dish according to claim 7, wherein: a liquid inlet (32) with a sealing cover is arranged between the rotating button (31) and the limiting block (33) and on the side wall of the penetrating part (35) in a penetrating way.
9. A method for testing bacteriostatic ability of bacteriostatic protein is characterized by comprising the following steps: the use of a culture dish according to any of claims 1 to 8 for testing, comprising the steps of:
(1) transferring the gene of the bacteriostatic protein into bacteria to be inhibited;
(2) adding culture media suitable for the growth of the bacteria in the step (1) into each interval of the culture dish plate (10), wherein the culture media at least comprise 1 liquid culture medium and 1 solid culture medium;
(3) inoculating the bacteria in the step (1) into the interval of the dish plate (10) filled with the liquid culture medium, and performing shake culture;
(4) adding bacteriostatic protein induction expression agent into liquid culture medium containing bacteria by using a pipette (30), and performing shake culture;
(5) sucking the contents cultured in the liquid culture medium in the step (4) by using a pipette (30), respectively inoculating the contents to the solid culture medium in other intervals of the dish plate (10), and continuously culturing;
(6) and observing the growth of bacteria on the solid culture medium in each interval.
10. A method of testing the bacteriostatic ability of a bacteriostatic protein according to claim 9, characterized in that: step (4) is performed during the log phase of bacterial growth.
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