CN109913442A - A kind of method of enteron aisle probiotics genetic modification - Google Patents
A kind of method of enteron aisle probiotics genetic modification Download PDFInfo
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Abstract
The present invention provides a kind of method of enteron aisle probiotics genetic modification.Method using synthetic biology will carry out microencapsulation processing after the primary bacillus coli gene transformation of the enteron aisle filtered out.By primary E. coli secretion simple function bioactive molecule --- for viscous protein (Ag43), it is studied in the application of inflammatory bowel disease (IBD) treatment method.A kind of new approaches are provided for research enteric microorganism and body interaction.The synthetic biology technology of bio-safety is combined into the treatment applied to intestines problem with hurtless measure formula light genetic technique, have many advantages for the method that traditional intestines problem is treated: the primary Escherichia coli of screening enteron aisle carry out genetic modification, improve the biological compliance of later period microencapsulation probiotics intestinal colonisation.Synthetic biology technology is combined with light genetic technique, realizes the accuracy controlling to the bioactive molecule for the treatment of intestines problem on space-time.
Description
Technical field
The present invention relates to microorganism and clinical treatment fields, and in particular to a kind of method of enteron aisle probiotics genetic modification.
Background technique
Enteron aisle as the maximum organ of organism, digest and assimilate, in terms of played important function.Enteron aisle
It is interior in addition to playing digestion and absorption, other than the epithelial cell of immunoloregulation function, immunocyte, further comprising many microorganisms.Its
In greater than 99.9% microorganism be all bacterium, they are referred to as intestinal flora.
" homobium " of the intestinal flora as organism enteron aisle, has participated in the regulation of many important life processes and disease.
The method of existing research enteric microorganism and body relationship is mostly that germfree mouse or extensive flora is utilized to transplant, in conjunction with microorganism
Group is learned and the analyses such as metabolism group, finds enteric microorganism for the regulation of disease and is accurate to molecule or gene target.But
Germfree mouse is at high price, and since there are certain immune deficiencies after gene knockout, cannot fully meet research needs.Bacterium
The problems such as group's transplanting is long in the presence of the transplanting period, is colonized low efficiency.
We filter out mouse intestinal fungal component E.coli NGF-1 from the angle of synthetic biology, utilize " light sensation
Promoter+functional molecular " mode (pDawn-GFP-Ag43, pDawn-RGF-TGF- β 1) is adhered using the regulation of light genetic approach
The expression of albumen and conversion growth factor, the fast, accurately field planting with realization to intestinal flora, and be with conversion growth factor
Example probes into influence of the enteric microorganism secretion single-element to body.It is used to be more suitable living body, the upper conversion of addition is micro-
The strong near infrared light of penetration into tissue is converted to visible light local in enteron aisle by ball, field planting or even micro- life to intestinal flora
Object plays facilitation to the research of body biological function.
Summary of the invention
The present invention in order to overcome the deficiencies of the prior art, provides a kind of method of enteron aisle probiotics genetic modification.
In order to solve the above technical problems, the technical scheme is that a kind of method of enteron aisle probiotics genetic modification, benefit
With the method for synthetic biology microencapsulation processing, key step will be carried out after the primary bacillus coli gene transformation of the enteron aisle filtered out
It is as follows:
1) screening of the primary Escherichia coli of C57BL/6 mouse intestinal；
2) genetic modification is carried out to primary Escherichia coli using the means of synthetic biology
(1) two sets of pUC pUCs: pDawn-GFP-Ag43 are synthesized using the means of synthetic biology, wherein pDawn is blue
Photoresponse promoter is section of DNA sequence.After receiving blue light signals, the transcription of downstream gene GFP-TGF- β 1 will start；
RFP translates into green fluorescent protein after transcription, serves as the function of reporter gene；Ag43 is viscous protein, is a kind of protide
Bioactive functions molecule；
(2) chemical induction-CaCl is utilized2Method induces the primary Escherichia coli filtered out at competence；
(3) plasmid conversion is carried out, light-operated pUC pUC is transformed into primary E. coli competent, by observing fluorescence
The expression of label expressed to prove functional molecular Ag43；
3) improved probiotics are subjected to microencapsulation processing.
Step 1) the specific steps are as follows:
(1) take the fresh intestinal tissue of C57BL/6 mouse in sterile EP tube；
(2) intestinal contents are rinsed with the sterile PBS being pre-chilled in advance；
(3) it after splitting intestinal walls, is cut to pieces with sterilized slide glass and takes inner wall of intestine, by inner wall of intestine object and sterile pre-cooling PBS
After mass volume ratio m/v=1:10 mixing, whirlpool shakes 15min；
(4) by 4 DEG C of mixture, 2000rpm is centrifuged 5min, discards precipitating, and by supernatant in 4 DEG C, 12000rpm is centrifuged
20min is discarded supernatant, and stays precipitating；
(5) after precipitating being resuspended with 100 μ L PBS, 10 μ L is taken to be coated on the solid LB plate for the screening of blue blank,
It 37 DEG C, is incubated overnight；
(6) second days growing states according to plate, picking blue colonies are used for bacterium colony PCR (two pairs of primers of bacterium colony PCR
LaZ-YZ-F:AACGGGGGTACTGACGAAAC respectively；LaZ-YZ-R:GAACCATCCGCTGTGGTACA), according to bacterium colony PCR
Purpose band size, filter out the primary Escherichia coli of C57BL/6 mouse intestinal.
Step 3) the specific steps are as follows:
(1) sterilization treatment is carried out after sodium alginate to be made into the solution of mass fraction 1-10%, by culture to logarithmic growth
The primary Escherichia coli of mid-term are added in the sodium alginate soln by sterilization treatment, are stirred, micro- as preparing
The interior liquid of capsule；
(2) external solution will be used as after dimethicone and CaCl2 admixture of powder；
The flow velocity of interior liquid syringe pump is 10uL/min, and the flow velocity of external solution syringe pump is 200uL/min.Syringe pump and opposite-flushing type
It is connected between capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop CaCO of generation3Acetum receive
Collection, the probiotics Microcapsules Size generated at this time are 500um.
Beneficial effects of the present invention:
1. the present invention is used for intestines problem treatment method, this is easy to operate, strong applicability, at low cost.And by " synthesising biological
- nanotechnology-light science of heredity " organically blends, and develops the efficiently convenient field planting of a set of achievable flora and can accurately control micro-
The nanometer light genetic system of the bio-safety of the single secretion element of biology.Compared to Oral Chemical drug therapy, microorganism is utilized
Treatment have it is more efficient, therapeutic effect is more preferable, polypeptide, protein medicaments especially suitable for half-life short.
2. for the method for traditional treatment IBD, by this treatment side IBD based on microencapsulation enteron aisle probiotics
Method has many advantages: being transformed to the primary Escherichia coli of the enteron aisle of screening, and it is fixed to improve later period microencapsulation probiotics enteron aisle
The biological compliance of plant.Synthetic biology technology is combined with light genetic technique, realizes the biology to treatment intestines problem
Accuracy controlling of the bioactive molecule on space-time.
3. of the invention innovative organically blends " synthetic biology-nanotechnology-light science of heredity ", develop it is a set of can
It realizes the efficiently convenient field planting of flora and can accurately control the nanometer light hereditary system of the bio-safety of the single secretion element of microorganism
System.
4. can be realized by means of the subsequent microfluidic platform built to micro-capsule using opposite-flushing type glass micro-fluidic device
Change the mass preparation of probiotics and upper conversion hydrogel microsphere.
5. accuracy of the light science of heredity on space-time is utilized this process employs the safe and efficient property of synthetic biology.
It for the method for traditional treatment IBD, improves patient and treats compliance, reduce the gastrointestinal tract thorn that conventional medicament is given
Swash.
6. providing a kind of new approaches for microorganisms and body interaction.
Detailed description of the invention
Fig. 1: according to the primer of design, the bacterium colony PCR band of the enteron aisle probiotics of screening；
Fig. 2: plasmid map pDawn-GFP-Ag43；
A.pDawn-Ag43 plasmid map
B.GFP plasmid map
Fig. 3: the visualization commensal gut bacterium for above converting microballoon guidance is colonized efficiency.
Specific embodiment
To further illustrate the present invention, now by specific implementation example, the present invention will be described in detail.
Embodiment 1
Method using synthetic biology will carry out at microencapsulation after the primary bacillus coli gene transformation of the enteron aisle filtered out
Reason:
1) screening of the primary Escherichia coli of C57BL/6 mouse intestinal
1. taking the fresh intestinal tissue of C57BL/6 mouse in sterile EP tube；
2. rinsing intestinal contents with the sterile PBS being pre-chilled in advance；
3. after intestinal walls are splitted, being cut to pieces with sterilized slide glass and taking inner wall of intestine, by inner wall of intestine object and sterile pre-cooling PBS matter
After measuring volume ratio m/v=1:10 mixing, whirlpool shakes 15min；
4. 2000rpm is centrifuged 5min by 4 DEG C of mixture, precipitating is discarded, by supernatant in 4 DEG C, 12000rpm is centrifuged 20min,
It discards supernatant, stays precipitating；
5. will precipitating with 100 μ L PBS be resuspended after, take 10 μ L be coated on for blue blank screen solid LB plate, 37
DEG C, it is incubated overnight；
6. second day growing state according to plate, picking blue colonies are used for bacterium colony PCR (two pairs of primers of bacterium colony PCR
It is LaZ-YZ-F:AACGGGGGTACTGACGAAAC respectively；LaZ-YZ-R:GAACCATCCGCTGTGGTACA), according to bacterium colony
The size of PCR purpose band filters out the primary Escherichia coli of C57BL/6 mouse intestinal.Wherein bacterium colony PCR band is shown in Fig. 1.
2) genetic modification is carried out to primary Escherichia coli using the means of synthetic biology
1. synthesizing a set of pUC pUC: pDawn-GFP-Ag43 using the means of synthetic biology, plasmid map is as schemed
2；
2. utilizing chemical induction-CaCl2Method induces the primary Escherichia coli filtered out at competence；
3. carrying out plasmid conversion, the light-operated plasmid of pDawn-GFP-Ag43 is transformed into primary E. coli competent, is led to
The expression of observation green fluorescence label is crossed to prove the expression of functional molecular (Ag43).
3) improved probiotics are subjected to microencapsulation processing
1. sterilization treatment is carried out after sodium alginate to be made into the solution of mass fraction 1%, by culture to mid log phase
Primary Escherichia coli be added in the sodium alginate soln by sterilization treatment, be stirred, as preparing microcapsules
Interior liquid；
2. by dimethicone and CaCl2External solution is used as after admixture of powder；
3. the flow velocity of interior liquid syringe pump is 10uL/min, the flow velocity of external solution syringe pump is 200uL/min.It syringe pump and liquidates
It is connected between formula capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop polytetrafluoroethylene (PTFE) culture of generation
Ware is collected, and the probiotics Microcapsules Size generated at this time is 500um.
Effect test:
The microencapsulation probiotics combination non-invasive light genetic technique being prepared is applied to the treatment of intestines problem.
(1) preparation of conversion light heredity micron order hydrogel particle on
1. being photoinitiator using PEGDA as crosslinking agent, HMPP, PEG and TE buffer is spackling, it is configured to pre-polymerization liquid,
Conversion particles in blue are added in pre-polymerization liquid, are stirred, as the interior liquid for preparing light heredity particle.Dimethyl-silicon
Oil is used as external solution；
2. the flow velocity of interior liquid syringe pump is 40uL/min, the flow velocity of external solution syringe pump is 800uL/min.It syringe pump and liquidates
It is connected between formula capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop polytetrafluoroethylene (PTFE) culture of generation
Ware is collected, and the upper hydrogel particle partial size of converting generated at this time is 500um.
(2) foundation of C57BL/6 mouse IBD model
Fasting for 24 hours, can't help water before C57BL/6 mouse IBD modeling.After weighing, 3% amobarbital intraperitoneal injection of anesthesia
(or ether), 5%2,4,6- trinitrobenzene sulfonic acid in 100mg/kg-150mg/kg ratio inject mouse intestinal.Mouse position
Upside down, to be inserted into anus upper end 4cm with PA tube, syringe injection is stood upside down three minutes or so, to prevent liquid medicine spill.Often
It is primary every three days bowel lavage, it can be made into IBD model mouse after 4 times.
(3) IBD is alleviated in field planting and combination non-invasive light genetic technique of the microencapsulation probiotics in C57BL/6 mouse intestinal
Mouse inflammation
After microencapsulation probiotics and upper conversion hydrogel particle are mixed, the mode of stomach-filling is taken to be injected into IBD model mouse
Alimentary canal in.After four hours, mouse anesthesia is handled, near infrared light pulsed exposure mouse peritoneal, Continuous irradiation one
Hour.Primary Escherichia coli can stablize field planting in mouse intestinal after Continuous irradiation seven days, play its corresponding function.Tissue is worn
After the strong near infrared light of permeability penetrates mouse peritoneal, conversion particles can be excited to issue blue light, the promoter impression of blue response
After blue light, start the expression of downstream gene, wherein function factor Ag43 assists field planting of the primary Escherichia coli in mouse intestinal.
For traditional bacterium solution stomach-filling mode, by means of function factor --- the extracellular expression of stickiness albumin A g43 significantly mentions
High field planting efficiency of the bacterial strain in mouse intestinal.
Embodiment 2
A kind of intestines problem treatment method of microencapsulation enteron aisle probiotics, the specific steps are as follows:
1) it will be carried out after the primary Escherichia coli progress genetic modification of the enteron aisle filtered out using the method for synthetic biology micro-
Encapsulated processing:
(1) screening of the primary Escherichia coli of C57BL/6 mouse intestinal
1. taking the fresh intestinal tissue of C57BL/6 mouse in sterile EP tube；
2. rinsing intestinal contents with the sterile PBS being pre-chilled in advance；
3. after intestinal walls are splitted, being cut to pieces with sterilized slide glass and taking inner wall of intestine, by inner wall of intestine object and sterile pre-cooling PBS matter
After measuring volume ratio m/v=1:10 mixing, whirlpool shakes 15min；
4. 2000rpm is centrifuged 5min by 4 DEG C of mixture, precipitating is discarded, by supernatant in 4 DEG C, 12000rpm is centrifuged 20min,
It discards supernatant, stays precipitating；
5. will precipitating with 100 μ L PBS be resuspended after, take 10 μ L be coated on for blue blank screen solid LB plate, 37
DEG C, it is incubated overnight；
6. second day growing state according to plate, picking blue colonies are used for bacterium colony PCR (two pairs of primers of bacterium colony PCR
It is LaZ-YZ-F:AACGGGGGTACTGACGAAAC respectively；LaZ-YZ-R:GAACCATCCGCTGTGGTACA), according to bacterium colony
The size of PCR purpose band filters out the primary Escherichia coli of C57BL/6 mouse intestinal.
(2) genetic modification is carried out to primary Escherichia coli using the means of synthetic biology
1. utilizing the means synthetic plasmid system of synthetic biology: pDawn-GFP-Ag43, plasmid map such as Fig. 2；
2. utilizing CaCl2Method induces the primary Escherichia coli filtered out at competence；
3. carrying out plasmid conversion, two sets of light-operated plasmids are transformed into primary E. coli competent, by observing fluorescence
The expression of label proves the expression of functional molecular (Ag4).
(3) improved probiotics are subjected to microencapsulation processing
1. sterilization treatment is carried out after sodium alginate to be made into the solution of mass fraction 10%, by culture to mid log phase
Primary Escherichia coli be added in the sodium alginate soln by sterilization treatment, be stirred, as preparing microcapsules
Interior liquid；
2. utilizing chemical induction-CaCl2Method induces the primary Escherichia coli filtered out at competence；
3. the flow velocity of interior liquid syringe pump is 10uL/min, the flow velocity of external solution syringe pump is 200uL/min.It syringe pump and liquidates
It is connected between formula capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop polytetrafluoroethylene (PTFE) culture of generation
Ware is collected, and the probiotics Microcapsules Size generated at this time is 500um.
Effect test: the microencapsulation probiotics combination non-invasive light genetic technique being prepared is applied to intestines problem
Treatment.
(1) preparation of conversion light heredity micron order hydrogel particle on
1. being photoinitiator using PEGDA as crosslinking agent, HMPP, PEG and TE buffer is spackling, it is configured to pre-polymerization liquid,
Conversion particles in blue are added in pre-polymerization liquid, are stirred, as the interior liquid for preparing light heredity particle.Dimethyl-silicon
Oil is used as external solution；
2. the flow velocity of interior liquid syringe pump is 40uL/min, the flow velocity of external solution syringe pump is 800uL/min.It syringe pump and liquidates
It is connected between formula capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop polytetrafluoroethylene (PTFE) culture of generation
Ware is collected, and the upper hydrogel particle partial size of converting generated at this time is 500um.
(2) foundation of C57BL/6 mouse IBD model
Fasting for 24 hours, can't help water before C57BL/6 mouse IBD modeling.After weighing, 3% amobarbital intraperitoneal injection of anesthesia
(or ether), 5%2,4,6- trinitrobenzene sulfonic acid in 100mg/kg-150mg/kg ratio inject mouse intestinal.Mouse position
Upside down, to be inserted into anus upper end 4cm with PA tube, syringe injection is stood upside down three minutes or so, to prevent liquid medicine spill.Often
It is primary every three days bowel lavage, it can be made into IBD model mouse after 4 times.
(4) IBD is alleviated in field planting and combination non-invasive light genetic technique of the microencapsulation probiotics in C57BL/6 mouse intestinal
Mouse inflammation
After microencapsulation probiotics and upper conversion hydrogel particle are mixed, the mode of stomach-filling is taken to be injected into IBD model mouse
Alimentary canal in.After four hours, mouse anesthesia is handled, near infrared light pulsed exposure mouse peritoneal, Continuous irradiation one
Hour.Primary Escherichia coli can stablize field planting in mouse intestinal after Continuous irradiation seven days, play its corresponding function.Tissue is worn
After the strong near infrared light of permeability penetrates mouse peritoneal, conversion particles can be excited to issue blue light, the promoter impression of blue response
After blue light, start the expression of downstream gene, wherein function factor Ag43 assists field planting of the primary Escherichia coli in mouse intestinal,
Function factor TGF-β 1 is used for the alleviation and treatment of IBD mouse inflammation.
Morphologic observation, particles size and distribution measurement:
Take microencapsulation probiotics and upper conversion hydrogel particle sample after centrifuge washing respectively,
Particle is put in liquid nitrogen after quick-frozen 4h, vacuum freeze drying is overnight, moisture in removal microcapsules and hydrogel, and one
Part is used to observe pattern, and after a part fixes particle with OCT embedding medium, frozen section observes its pattern under scanning electron microscope
State is simultaneously taken pictures.Observe microencapsulation probiotics and upper conversion hydrogel particle in the spherical grain of uniformly rule under transmission electron microscope
Son, microencapsulation probiotics and upper conversion hydrogel particle internal structure are loose, can significantly embed bacterium and upper conversion particles.And
It can control two microencapsulation probiotics of synthesis and the partial size of upper conversion hydrogel particle by the flow velocity of syringe pump.
Mouse Digestive is injected by way of stomach-filling after microencapsulation probiotics and upper conversion hydrogel particle are mixed
In system.Experimental mice: it by after mouse anesthesia after four hours, with pulsed near infrared light mouse peritoneal 1 hour, continuously shines
It penetrates 7 days；Control group mice: without any processing after stomach-filling.Mouse is dissected in batches after seven days, and intestinal contents is taken to carry out streaming
Cell instrument detection.The distribution rolled into a ball by detection bacterium, to determine that the number of the primary Escherichia coli of C57BL/6 mouse intestinal is arrived in field planting
Amount.
Conversion light heredity micron order hydrogel and microencapsulation probiotics, are innovated first on above-described embodiment 1-2 products therefrom
Conversion light heredity micron order hydrogel in the synthesis of property, is prepared for the upper conversion particles of uniform particle diameter；Secondly innovative synthesis
Microencapsulation probiotics, the preparation room of two of them particle are erected micro-fluidic flat by opposite-flushing type glass micro-fluidic device
Platform simplifies the preparation flow of preparation two kinds of particles.Finally be field planting of the microorganisms in enteron aisle, have it is great theoretical and
Realistic meaning.Synthetic biology, light science of heredity combined with nanobiology, design construction has safe and accurate, noninvasive
Nano-hydrogel system, effectively improve intestines bacterium transplanting metabolism controllability, finally for intestines bacterium transplanting be used for disease prevention and cure, provide
A kind of new method.
The present invention has the advantage that compared with the Oral Chemical drug of traditional treatment intestines problem
To sum up, a kind of microencapsulation probiotics of transformation of the present invention and in terms of intestines problem treatment application include with
Under several aspects: 1) screening of the primary Escherichia coli of C57BL/6 mouse intestinal；2) using the means of synthetic biology to primary big
Enterobacteria carries out genetic modification；3) improved probiotics are subjected to microencapsulation processing；4) conversion light heredity micron order water-setting on
The preparation of glue particle；5) foundation of C57BL/6 mouse IBD model；6) microencapsulation probiotics are determined in C57BL/6 mouse intestinal
It plants and non-invasive light genetic technique is combined to alleviate IBD mouse inflammation.
The microencapsulation probiotics of this transformation are especially led in intestinal flora and body interaction in intestines problem therapy field
Under domain novelty application principle is: 1) conversion light heredity micron order hydrogel joint microencapsulation probiotics on, by synthetic biology,
Light science of heredity is combined with nanobiology, and design construction is with safe and accurate, non-invasive characteristic nano-hydrogel system.
In addition the primary Escherichia coli of synthetic biology transformation have sensitive blue response, the accurate table for controlling downstream functional protein
It reaches；2) conversion light heredity micron order hydrogel joint microencapsulation probiotics are treated applied to intestines problem on, initiative by light
Genetic technique applies to the treatment of intestines problem, for the relationship between research body intestinal flora and disease, provides a kind of complete
New thinking.
Upper conversion light heredity micron order hydrogel and microencapsulation probiotics main performance index include: a) partial size 500nm
Upper conversion light heredity micron order hydrogel and microencapsulation probiotics, partial size can inject flow rate pump etc. according to the constituent of preparation
Be adjusted, uniform particle diameter, stable appearance and have visual micron scale construction；B) whole preparation process is simple and fast,
Short preparation period, yield is high, is suitble to produce in enormous quantities；C) conversion light heredity micron order hydrogel joint microencapsulation probiotics are answered on
It is treated for intestines problem, using the expression of light genetic approach regulation Fibronectin, to realize to the quick, accurate of intestinal flora
Field planting provides a kind of new approaches to probe into influence of the enteric microorganism secretion single-element to body.In Fig. 3, in light heredity
In regulation method and conventional method, by the expression of cell streaming examining report gene, microorganism can be realized in enteron aisle
Field planting, but two methods are compared, light science of heredity regulates and controls method while improving Microorganism colonization efficiency, and it is fixed to substantially reduce
Plant the period.It is the new method of a kind of efficient, quick, convenient and fast microorganisms and body interaction.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (3)
1. a kind of method of enteron aisle probiotics genetic modification, it is characterised in that: the method using synthetic biology will filter out
Microencapsulation processing is carried out after the primary bacillus coli gene transformation of enteron aisle, key step is as follows:
1) screening of the primary Escherichia coli of C57BL/6 mouse intestinal；
2) genetic modification is carried out to primary Escherichia coli using the means of synthetic biology
(1) utilize the means synthetic plasmid system of synthetic biology: pDawn-GFP-Ag43, wherein pDawn is that blue response is opened
Mover will start the transcription of downstream gene GFP-Ag43 after receiving blue light signals；
(2) chemical induction-CaCl is utilized2Method induces the primary Escherichia coli filtered out at competence；
(3) plasmid conversion is carried out, light-operated pUC pUC is transformed into primary E. coli competent, by observing fluorescence labels
Expression prove the expression of functional molecular Ag43；
3) improved probiotics are subjected to microencapsulation processing.
2. a kind of method of enteron aisle probiotics genetic modification according to claim 1, it is characterised in that: the step 1) tool
Body step are as follows:
(1) take the fresh intestinal tissue of C57BL/6 mouse in sterile EP tube；
(2) intestinal contents are rinsed with the sterile PBS being pre-chilled in advance；
(3) it after splitting intestinal walls, is cut to pieces with sterilized slide glass and takes inner wall of intestine, by inner wall of intestine object and sterile pre-cooling PBS mass
After volume ratio m/v=1:10 mixing, whirlpool shakes 15min；
(4) by 4 DEG C of mixture, 2000rpm is centrifuged 5min, discards precipitating, and by supernatant in 4 DEG C, 12000rpm is centrifuged 20min, abandons
Supernatant is removed, precipitating is stayed；
(5) will precipitating with 100 μ L PBS be resuspended after, take 10 μ L be coated on for blue blank screen solid LB plate, 37 DEG C,
It is incubated overnight；
(6) second days growing states according to plate, picking blue colonies for bacterium colony PCR, (distinguish by two pairs of primers of bacterium colony PCR
LaZ-YZ-F:AACGGGGGTACTGACGAAAC；LaZ-YZ-R:GAACCATCCGCTGTGGTACA), according to the mesh of bacterium colony PCR
Stripe size, filter out the primary Escherichia coli of C57BL/6 mouse intestinal.
3. a kind of method of enteron aisle probiotics genetic modification according to claim 1, it is characterised in that: the step 3) tool
Body step are as follows:
(1) sterilization treatment is carried out after sodium alginate to be made into the solution of mass fraction 1-10%, by culture to mid log phase
Primary Escherichia coli be added in the sodium alginate soln by sterilization treatment, be stirred, as preparing microcapsules
Interior liquid；
(2) by dimethicone and CaCl2External solution is used as after admixture of powder；
(3) flow velocity of interior liquid syringe pump is 10uL/min, and the flow velocity of external solution syringe pump is 200uL/min.Syringe pump and opposite-flushing type
It is connected between capillary microlayer model chip with PE pipe, that is, builds microfluidic platform, the drop CaCO of generation3Acetum receive
Collection, the probiotics Microcapsules Size generated at this time are 500um.
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