CN107460117A - A kind of experimental provision and method for resistant gene Horizontal Gene Transfer research - Google Patents
A kind of experimental provision and method for resistant gene Horizontal Gene Transfer research Download PDFInfo
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- CN107460117A CN107460117A CN201710786877.1A CN201710786877A CN107460117A CN 107460117 A CN107460117 A CN 107460117A CN 201710786877 A CN201710786877 A CN 201710786877A CN 107460117 A CN107460117 A CN 107460117A
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- blake bottle
- filter membrane
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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
- C12M23/08—Flask, bottle or test tube
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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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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Abstract
A kind of experimental provision and method for resistant gene Horizontal Gene Transfer research, agent structure is using the triangle blake bottle of Liang Ge UNICOMs as carrier, it is connected in the upper left of triangle blake bottle provided with thin neck with vavuum pump, bottom right is provided with connector in case connection between two blake bottles, two glass blake bottles are connected by flange, filter membrane is provided between flange, to prevent the direct contact of cell but without prejudice to other mass exchanges, filter membrane is attached among core sieve plate, it is separated by between sieve plate and the blake bottle mouth of pipe with silica gel pad to seal, above material is put into flange male-pipe again with the connection sealing of internal thread side in order.Heretofore described glass blake bottle, core sieve plate, silica gel pad and composite fibre filter membrane can high-temperature sterilization, to exclude living contaminants.Simple in construction, convenient experimental operation, filter membrane can be changed at any time, and core can play a part of protecting filter membrane as holder, and cell can be prevented directly to contact and will not be damaged because of vacuum filtration.
Description
Technical field
The propagation Pollution Study of present invention antibiotics resistance gene suitable for environment, more particularly to resistant gene is in environment
In risk investigation of the engagement transfer in gene transfer, be a kind of experiment dress for resistant gene Horizontal Gene Transfer research
Put and method.
Background technology
2006, Pruden et al. proposed antibiotics resistance gene (Antibiotic Resistance first
Genes, ARGs) be used as the emerging pollutant of a kind of environment, ARGs unlike other pollutants has heredity, can parental generation with
Transmitted between filial generation by way of vertically shifting, can also be by moveable genetic elements horizontal transfer to not having originally
ARGs thalline.Therefore, long-term and irreversible influence can be caused on environment.If human poultry infection carries ARGs's
Pathogenic bacteria, during treatment the effect of antibiotic will substantially reduce, increase the difficulty of healing.Research shows to be used for medical treatment or animal
The antibiotic of cultivation there are about 75%-95% with active compound or metabolite form discharge body, frequently using and arranging due to antibiotic
Put, form the accumulation of certain level in the environment, and selection pressure is formd to antibiotic resistance, and then promote ARGs's
Propagate and convert.Generally in surrounding medium, bacterial strain by Horizontal Gene Transfer (Horizontal Gene Transfer,
HGT mode) obtains antibiotic resistance.Because HGT can be transmitted between of the same race or different genera bacterial strain, thus greatly
The big appearance for accelerating resistant gene.Conversion, transduction and engagement are considered as the major way of Horizontal transfer.Wherein engage
Transfer relies on the direct contact between microorganism, and nineteen fifty Bernard D.Davis proposes the glass tube pair of the connection with filter plate
The mating experiment of deficient strain also demonstrates this point, but to the experiment in Bernard D.Davis original
Only simply refer to, do not clearly state the details of experiment.
The content of the invention
The shortcomings that in order to overcome above-mentioned prior art, it is an object of the invention to provide one kind to be used for the horizontal base of resistant gene
Because of the experimental provision and method of transfer research, device is designed primarily to the triangle blake bottle of Liang Ge UNICOMs, be easy to put, operate and
Sampling, two triangle blake bottles are connected by flange, are readily disassembled, sample, clean and control, and are provided among flange replaceable
Filter membrane, filter sizes are easily controllable also can be needed to set different apertures according to experiment.The present invention reaches practicality simple to operate
Have the function that the direct contact between control engagement transfer cell again, and it is simple for production, and can sterilize reuse.
To achieve these goals, the technical solution adopted by the present invention is：
A kind of experimental provision for resistant gene Horizontal Gene Transfer research, trained including at least two triangles that can be sterilized
Support bottle, it is characterised in that be provided with the triangle blake bottle and the thin eck of pump be filtered by vacuum for connecting and for other
The connector of triangle blake bottle connection, is connected at the connector by flange, be provided among flange can prevent cell it
Between direct contact but without prejudice to other mass exchanges between the triangle blake bottle being connected filter membrane.
The thin eck is arranged on the top of the connector, and the thin eck is located at triangle respectively with the connector
In the relative both direction of blake bottle, obliquely, the connector is horizontal connection mouth to the thin eck opening.
The triangle blake bottle is connected two-by-two by connector, i.e. a triangle blake bottle only with an adjacent triangle
Blake bottle is connected by connector.
The triangle blake bottle connects vacuum filtration pump by thin eck, is trained by being filtered by vacuum the triangle being connected
Mass exchange occurs between foster bottle.
Using core sieve plate as holder, the core sieve plate is overlying on connection with the silica gel pad of annular for the filter membrane both sides
In pipe, the core sieve plate and silica gel pad are supporting and protect filter membrane.
The flange is divided into internal thread side and external screw thread side, is respectively placed in the connector for the triangle blake bottle being connected
Place, fixed with polyethylene ring, the filter membrane, core sieve plate and silica gel pad size are consistent, with silica gel pad, core sieve plate,
Filter membrane, core sieve plate, the order of silica gel pad load flange external screw thread side after stacking, by external screw thread side and internal thread side
Connect and seal, to change filter membrane at any time, or the filter membrane of selection different pore size size is needed according to experiment.
The volume of the triangle blake bottle is 250ml；The core hole diameter of sieve (perforated) plate is 10 μm；The connector is a diameter of
3cm；The filter sizes are 0.22 μm.
The filter membrane can be cellulose mixture filter membrane.
The filter membrane is located between the pipe of experimental provision two.
Present invention also offers the experimental method for resistant gene Horizontal Gene Transfer research, can be gone out using at least two
The triangle blake bottle of bacterium, thin eck connection vacuum filtration pump is set, by each triangle blake bottle and its on each triangle blake bottle
Its triangle blake bottle is connected by connector, and filter membrane is set on connecting line, by being filtered by vacuum be connected three
Mass exchange occurs between the blake bottle of angle, the direct contact between cell is prevented by filter membrane.
Compared with prior art, the beneficial effects of the invention are as follows：Cell can be prevented directly to contact but do not influence to include disease
The exchange of other macromoleculars such as poison, plasmid or small-molecule substance, therefore can be used for horizontal turn of antibiotics resistance gene in environment
The research of shifting.Special engagement transfer needs the direct contact between cell, and the present invention can exclude conversion caused by plasmid and make
With with virus caused by transduction, for resistant gene engagement transfer research new Research Thinking is provided.
Brief description of the drawings
Fig. 1 is the experimental provision schematic diagram of the present invention.
Fig. 2 is flange internal structure schematic diagram.
In figure, 1 is triangle blake bottle, and 2 are filtered by vacuum the thin eck of pump for connection, and 3 be the connection of two triangle blake bottles
Mouthful, 4 be flange, and 5 be silica gel pad, and 6 be core sieve plate, and 7 be filter membrane.
Fig. 3 is that each processing detects fluorescence signal expression of results for acceptor laser confocal microscope in the embodiment of the present invention.
Wherein, a represents Escherichia coli, and b represents pseudomonas putida, and c represents blake bottle sample on the right side of experimental provision, d generations
Table triangle blake bottle mixed culture.
Embodiment
Describe embodiments of the present invention in detail with reference to the accompanying drawings and examples.
Embodiment 1
By plasmid, diffusion experiment proves that the present invention does not influence plasmid, bacteriophage etc. in two triangle cultures in experimental provision
Exchange between bottle.
As shown in figure 1, in device, triangle blake bottle 1 has two, and using glass material, the upper left corner of triangle blake bottle 1 is opened
There is thin eck 2, the lower right corner is provided with connector 3, and silica gel pad 5 is annular, and the specification of filter membrane 7 is 0.22 μm.
Two triangle blake bottles 1, core sieve plate 6, silica gel pad 5 and the high-temperature sterilization of filter membrane 7, flange 4 are passed through into 75% wine
Essence sterilization is simultaneously degerming by ultraviolet irradiation, experimental provision is connected standby.
As shown in Fig. 2 connected mode is：
The thin connection of eck 2 vacuum filtration pump, flange 4 are arranged at the connector 3 of two triangle blake bottles 1, and flange 4, which divides, is
Internal thread side and external screw thread side, are respectively placed at the connector 3 of two triangle blake bottles 1, are fixed with polyethylene ring, with silicon
Glue gasket 5, core sieve plate 6, filter membrane 7, core sieve plate 6, the order of silica gel pad 5 load flange external screw thread side after stacking, will
External screw thread side is connected and sealed with internal thread side.
Using copy number as 6.35 × 109Copies/ μ l standard r plasmid sul1 is experiment material, is diluted with aqua sterilisa
Into 6.35 × 105copies/μl.150ml 6.35 × 10 will be poured into the left blake bottle of experimental provision9Copies/ μ l plasmids are molten
Liquid, right blake bottle pour into 150ml aqua sterilisas, aspirate 2 bouts back and forth using pump is filtered by vacuum, respectively 0,15 after inoculation,
200 μ l are sampled in 30,60min or so two blake bottles, are surveyed using sample during quantitative fluorescent PCR measure 0,15,30,60min
Determine sul1 concentration.
Quantitative (the copies/ μ l) test results of the plasmid sul1 of table 1
Experimental result illustrates that device is without prejudice to the mass exchange in addition to cell in the present invention, therefore the plasmid in left side can
To reach balance on the right side of free enter and after 60min.
Embodiment 2
Prove that the present invention can prevent cell by shift experiment of the resistance bacterium of fluorescent protein labeling in experimental provision
Between direct contact.
It is consistent with embodiment 1 using device.
It is donor bacterium with the Escherichia coli ATCC25922 of antibiotic sensitive using the pseudomonas putida KT2400 of fluorescence labeling
For recipient bacterium, pseudomonas putida KT2400 carries the broad host range plasmid RP4 that marked green fluorescent protein, and the bacterium is dyed
Body then marked red fluorescent protein and lacIq genes, can express red fluorescence but suppress the expression of plasmid green fluorescent protein,
To be shifted if there occurs engagement, plasmid RP4 will be transferred to recipient bacterium and fluoresced green.Will be for acceptor respectively at fluid nutrient medium
Interior 30 DEG C, 160r/min is incubated overnight.Then the culture of two blake bottles in left and right of experimental provision is inoculated in 1% inoculum concentration
In base (150ml), and two bacterium are mixed in 250ml triangle blake bottles as compareing, respectively 0,15 after inoculation,
30,60min sample 200 μ l in U-shaped experimental provision and triangle blake bottle, remember Uz, Uy, Mating.Wherein 100 μ l are used to be coated with
Resistant panel, 100 μ l are used for the luciferase expression of laser confocal microscope detection sample.
The sample resistance count results of table 2
Donor bacterium used in the present invention can grow in resistant panel, and recipient bacterium in resistant panel without growth.Reference picture
3, by Olympus laser confocal microscope FV3000 detect sample luciferase expression, using recipient bacterium ATCC25932 as
There is no the negative control (Fig. 3 a) of fluorescence, (Fig. 3 b, marked using donor bacterium KT2440 as red fluorescence positive control with circle),
The wide spectrum that excites for selecting fluorescence is respectively 488nm and 561nm, takes the μ l of sample 100 during each experiment process 60min to be placed in copolymerization burnt
In capsule, identical zoom values detection luciferase expression is selected.Sample (Fig. 3 c), has no luciferase expression on the right side of Fig. 3 c experimental provisions, and
The triangular flask (Fig. 3 d) of mixed culture then detects the joint element (with square marks) of green fluorescence, further illustrates device resistance
Donor bacterium is stopped and has entered right-hand member from left end, hindered the direct contact of cell, available for antibiotic resistance strain resistant gene
Horizontal transfer.
Claims (9)
- A kind of 1. experimental provision for resistant gene Horizontal Gene Transfer research, including at least two triangle that can be sterilized cultures Bottle, it is characterised in that be provided with the triangle blake bottle and the thin eck of pump be filtered by vacuum for connecting and for other three The connector of angle blake bottle connection, is connected at the connector by flange, and being provided among flange can prevent between cell Direct contact but without prejudice to other mass exchanges between the triangle blake bottle being connected filter membrane.
- 2. it is used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1, it is characterised in that described Thin eck is arranged on the top of the connector, and the thin eck is located at the relative of triangle blake bottle respectively with the connector In both direction, obliquely, the connector is horizontal connection mouth to the thin eck opening.
- 3. it is used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1, it is characterised in that described Triangle blake bottle is connected two-by-two by connector, i.e. a triangle blake bottle only passes through company with an adjacent triangle blake bottle Interface connects.
- 4. it is used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1, it is characterised in that described Triangle blake bottle connects vacuum filtration pump by thin eck, is occurred by being filtered by vacuum between the triangle blake bottle being connected Mass exchange.
- 5. it is used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1, it is characterised in that described Filter membrane both sides are using core sieve plate as holder, and the core sieve plate is overlying in connecting tube with the silica gel pad of annular, the sand Core sieve plate and silica gel pad are supporting and protect filter membrane.
- 6. it is used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 5, it is characterised in that described Flange is divided into internal thread side and external screw thread side, is respectively placed at the connector for the triangle blake bottle being connected, with polyethylene Ring is fixed, and the filter membrane, core sieve plate and silica gel pad size are consistent, is sieved with silica gel pad, core sieve plate, filter membrane, core Plate, the order of silica gel pad load flange external screw thread side after stacking, and external screw thread side is connected and sealed with internal thread side, To change filter membrane at any time, or the filter membrane of selection different pore size size is needed according to experiment.
- 7. being used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1 or 5 or 6, its feature exists In the volume of the triangle blake bottle is 250ml；The core hole diameter of sieve (perforated) plate is 10 μm；The a diameter of 3cm of connector；Institute Filter sizes are stated as 0.22 μm.
- 8. being used for the experimental provision of resistant gene Horizontal Gene Transfer research according to claim 1 or 5 or 6, its feature exists In the filter membrane is cellulose mixture filter membrane.
- 9. the experimental method for resistant gene Horizontal Gene Transfer research, it is characterised in that can be sterilized using at least two Triangle blake bottle, thin eck connection vacuum filtration pump is set, by each triangle blake bottle and other three on each triangle blake bottle Angle blake bottle is connected by connector, and filter membrane is set on connecting line, is trained by being filtered by vacuum the triangle being connected Mass exchange occurs between foster bottle, passes through the direct contact between filter membrane prevention cell.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710786877.1A CN107460117A (en) | 2017-09-04 | 2017-09-04 | A kind of experimental provision and method for resistant gene Horizontal Gene Transfer research |
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| CN201710786877.1A CN107460117A (en) | 2017-09-04 | 2017-09-04 | A kind of experimental provision and method for resistant gene Horizontal Gene Transfer research |
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| CN107460117A true CN107460117A (en) | 2017-12-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317715A (en) * | 2019-08-16 | 2019-10-11 | 贵州大学 | A kind of mixed fermentation device for laboratory liquid state fermentation |
| CN114525198A (en) * | 2022-01-21 | 2022-05-24 | 江苏海洋大学 | Microalgae non-contact co-culture device |
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|---|---|---|---|---|
| CN201071368Y (en) * | 2007-08-10 | 2008-06-11 | 武汉大学 | U shaped pipe microbiology experimental device with filter film |
| CN104094901A (en) * | 2014-07-11 | 2014-10-15 | 江苏农牧科技职业学院 | Indoor high-density rotifer culture harvest method applicable to molecular biology studies |
| CN104894701A (en) * | 2015-06-25 | 2015-09-09 | 安徽华茂纺织股份有限公司 | Yarn end breakage preventing feeding device special for doubling winder |
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2017
- 2017-09-04 CN CN201710786877.1A patent/CN107460117A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201071368Y (en) * | 2007-08-10 | 2008-06-11 | 武汉大学 | U shaped pipe microbiology experimental device with filter film |
| CN104094901A (en) * | 2014-07-11 | 2014-10-15 | 江苏农牧科技职业学院 | Indoor high-density rotifer culture harvest method applicable to molecular biology studies |
| CN104894701A (en) * | 2015-06-25 | 2015-09-09 | 安徽华茂纺织股份有限公司 | Yarn end breakage preventing feeding device special for doubling winder |
Non-Patent Citations (1)
| Title |
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| 周文敏等: "《水和有害废物的监测分析方法》", 31 December 1990 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317715A (en) * | 2019-08-16 | 2019-10-11 | 贵州大学 | A kind of mixed fermentation device for laboratory liquid state fermentation |
| CN114525198A (en) * | 2022-01-21 | 2022-05-24 | 江苏海洋大学 | Microalgae non-contact co-culture device |
| CN114525198B (en) * | 2022-01-21 | 2022-12-06 | 江苏海洋大学 | Microalgae non-contact co-culture device |
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Application publication date: 20171212 |