CN109897863A - A kind of method for transformation of Lactobacillus brevis - Google Patents
A kind of method for transformation of Lactobacillus brevis Download PDFInfo
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- CN109897863A CN109897863A CN201910328711.4A CN201910328711A CN109897863A CN 109897863 A CN109897863 A CN 109897863A CN 201910328711 A CN201910328711 A CN 201910328711A CN 109897863 A CN109897863 A CN 109897863A
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- lactobacillus brevis
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Abstract
The invention discloses a kind of method for transformation of Lactobacillus brevis, belong to food microorganisms technical field.Since the biochemical characteristic of bacterial strain, molecule are constituted between lactic acid bacteria different genera and immunological characteristic is all different, the present invention is improved in existing Lactococcus lactis and without endogenous plasmid Lactobacillus brevis electrotransformation method on the basis of, Lactobacillus brevis is continuously activated twice with MRS culture medium with the MRS culture medium containing 1% glycine respectively, continue to collect and prepare competent cell when culture reaches higher concentration to thallus, to increase competent cell concentration and activity.The condition that shocks by electricity when electrotransformation exogenous dna fragment is 2.4KV, 3.5ms, increases electrotransformation efficiency.Through examining, foreign gene successful conversion competent cell solves the problems, such as the preparation of wild type Lactobacillus brevis electrotransformation competence and conversion.
Description
Technical field
The present invention relates to a kind of method for transformation of Lactobacillus brevis, belong to food microorganisms technical field.
Background technique
Lactic acid bacteria is that one kind generates the by-products such as lactic acid, ethyl alcohol, carbon dioxide in a manner of homofermentation or heterofermentation
Gram-positive bacterium general name, food industry such as grape wine, soy sauce, sausage fermented food and alcoholic beverage production in
Important beneficial effect is played, pH value, holding are such as adjusted and improves brewing micro-ecological environment, promotes saccharification and fermentation, promotes wind
Taste substance and its precursor generation etc..Wherein, mainly with lactobacillus plantarum, Lactobacillus brevis, lactobacillus fermenti etc. for dominant bacteria
Kind.But the metabolite that some toxic effects can be also generated after lactobacillus-fermented, as D-ALPHA-Hydroxypropionic acid, nitrite, alkamines,
Indoles etc., if this substance is accumulated excessively in livestock and poultry and human body, it will cause different diseases even cancer.
The development of Protocols in Molecular Biology facilitate the mankind inherently understand the evolution of lactic acid bacteria, functional characteristics and its with
Relationship between environment, and then apply lactic acid bacteria rationally in food and feedstuff industry.Although having tens of kinds so far
The full-length genome of lactic acid bacteria obtains sequencing data, but wherein there is the gene of many unknown functions, in spite of when show certain
The activity of a little enzymes, but its complete physiological function not easy to identify.Therefore, the specific metabolism of lactic acid bacteria is disclosed from gene level
Mechanism helps speed up strain breeding thereof and transformation, optimizes food, feed fermentation condition, realizes the safety in production of lactic acid bacteria, make work
The risk of industry lactic acid bacteria is reduced to zero.
Since lactic acid bacteria kind is more, the biochemical characteristic of bacterial strain between different genera, molecule constitute and immunological characteristic not phase
Together, different hosts bacterial strain needs successfully construct recombination engineering using different exogenous genetic fragment method for transformation.Mesh
Before, only a small number of kinds such as Lactococcus lactis, lactobacillus plantarum have mature method for transformation.2016, Yang Juan et al. was with plasmid
PMG36e, Pnz8148, pNZ9530 and Lactobacillus brevis CGMCC1306 without indigenous plasmid are material, by Lactobacillus brevis sense
The investigation of each influence factor, establishes a kind of electrotransformation experiment condition (bibliography: poplar during by state preparation and electrotransformation
Tiny stream, Lv Changjiang, Luo Maiqi wait mono- plant of electrotransformation condition [J] the food without indigenous plasmid Lactobacillus brevis and biotechnology journal,
2016,35(6):584-590).However, verifying through inventor, which is not suitable for other Lactobacillus brevis.Therefore,
A kind of scope of application is provided compared with the wide and higher Lactobacillus brevis method for transformation of efficiency, is had for the further application of Lactobacillus brevis
Important application value.
Summary of the invention
An object of the present invention is to provide a kind of Lactobacillus brevis method for transformation, which comprises
(1) Lactobacillus brevis is activated；
(2) Lactobacillus brevis after activating is cultivated to OD600For 0.8-1.0；
(3) thallus is collected, competent cell is prepared；
(4) it is added in competent cell after exogenous dna fragment through electric shock, electrotransformation Lactobacillus brevis competent cell, voltage
It is set as 2.4-2.6KV, the electric shock time is 2-4ms；
(5) competent cell is recovered, and is coated with MRS plate, screens transformant；
(6) transformant plasmid is extracted, and PCR is carried out according to exogenous dna fragment special primer, is expanded and is produced by electrophoresis detection
Object further verifies positive transformant.
In one embodiment of the invention, Lactobacillus brevis include Lactobacillus brevis 2-34, Lactobacillus brevis ATCC 367 or
Lactobacillus brevis TMW 1.2112.
In one embodiment of the invention, step (1) specifically: be inoculated in Lactobacillus brevis by 1-5% inoculum concentration
The activation of MRS fluid nutrient medium, 35-38 DEG C of stationary culture 10-14h；Culture solution is transferred in containing 1% sweet ammonia by 1-5% inoculum concentration
The MRS fluid nutrient medium of acid, 35-38 DEG C is continued to activate 20-30h.
In one embodiment of the invention, step (2) specifically: by 1-5% inoculum concentration by the short newborn bar after activation
Bacterium is inoculated in the MRS fluid nutrient medium containing 1% glycine, 35-38 DEG C of stationary culture to OD600For 0.8-1.0.
In one embodiment of the invention, step (3) specifically: the thallus for obtaining step (2) is centrifuged, and collects bacterium
It is thin to get competence that thallus is resuspended with Electroporation Buffer with Washing Buffer washing thalline cell in body
Born of the same parents.
In one embodiment of the invention, step (4) described electric-shocking method is specifically: taking exogenous plasmid dna and sense
In standing 5min on ice after being mixed by state cell, electric shock cup is moved into, voltage is set as 2.4-2.6KV, and the electric shock time is 2-4ms,
It is transformed into exogenous plasmid dna in competent cell.
Further, voltage is set as 2.4KV, and the electric shock time is 3.5ms.
In one embodiment of the invention, the ratio of exogenous plasmid dna and competent cell is 400-500:50
(ng/μL)。
In one embodiment of the invention, step (5) specifically includes: being added immediately into electric shock cup after electric shock
Cell is resuspended and is transferred to new centrifuge tube by MRS fluid nutrient medium, in standing 5-10min on ice, in 35-38 DEG C of incubator
Middle stationary culture 1.5-2h recovers, and is centrifuged bacterium solution, abandons part supernatant, and thallus is coated on MRS solid medium after being resuspended,
35-38 DEG C of stationary culture 4-5d.
In one embodiment of the invention, the Washing Buffer are as follows: 0.5mol/L sucrose, 0.5mmol/L
MgCl2,5mmol/L KH2PO4, tune pH are 7.4-7.5,115 DEG C of high pressure sterilization 20min.
In one embodiment of the invention, the Electroporation Buffer are as follows: 0.5mol/L sucrose,
115 DEG C of high pressure sterilization 20min.
It is a further object to provide the above methods in production Lactobacillus brevis intrinsic protein or foreign protein
Using.
Beneficial effects of the present invention:
The present invention provides a kind of method for transformation of Lactobacillus brevis, in existing Lactococcus lactis and are free of endogenous plasmid
It is improved on the basis of Lactobacillus brevis electrotransformation method, Lactobacillus brevis is used to MRS culture medium respectively and containing 1% glycine
MRS culture medium continuously activates twice, continues to collect and prepare competent cell when culture reaches higher concentration to thallus, to increase
Competent cell concentration and activity.Will when electrotransformation exogenous dna fragment shock by electricity condition by 2.0KV, 3.5ms be changed to 2.4KV,
3.5ms increases electrotransformation efficiency.Through examining, foreign gene successful conversion competent cell solves wild type Lactobacillus brevis electricity
The problem of transformed competence colibacillus preparation and conversion.
Detailed description of the invention
Fig. 1: the positive transformant pcr amplification product agarose gel electrophoretogram in embodiment 1.
Fig. 2: the positive transformant pcr amplification product agarose gel electrophoretogram in embodiment 2.
Fig. 3: the positive transformant pcr amplification product agarose gel electrophoretogram in embodiment 3.
Specific embodiment
Experimental material involved by following embodiment is as follows:
Lactobacillus brevis: used Lactobacillus brevis 2-34 (Lactobacillus brevis 2-34) is this in embodiment
Seminar from before yellow rice wine in ferment fermentation liquid it is separating obtained, be preserved in China typical culture collection on March 23rd, 2017
The heart, preservation address are Wuhan, China university, and deposit number is CCTCC NO:M 2017140, and the bacterial strain is in Publication No.
It is disclosed in the patent application of CN106978371A.
Reagent and kit: beef extract, sodium chloride, agar powder, citric acid, K2HPO4﹒ 3H2O、MgSO4﹒ 7H2O、MnSO4﹒
H2O, Tween-80, sodium acetate, DEXTROSE ANHYDROUS, acetic acid, sucrose, MgCl2Equal reagents are purchased from the limited public affairs of Chinese medicines group chemical reagent
Department.Peptone, yeast powder are purchased from Oxoid company.DNA Marker, PrimeStar Max Premix 2 ×, agarose is purchased from
Shanghai Sheng Gong bioengineering Co., Ltd.Gold-view nucleic acid dye is purchased from Beijing SBS Genetech Co., Ltd.Plasmid extracts reagent
Box E.Z.N.A.Plasmid DNA Mini Kit I D6943-02 200preps V-Spin is purchased from OMEGA company.
Washing Buffer:0.5mol/L sucrose, 0.5mmol/L MgCl2,5mmol/L KH2PO4 adjust pH=7.4,
115 DEG C of high pressure sterilization 20min.
Electroporation Buffer:0.5mol/L sucrose, 115 DEG C of high pressure sterilization 20min.
1 plasmid pNZ5319 of embodiment converts Lactobacillus brevis 2-34
(1) plasmid pNZ5319 electrotransformation Lactobacillus brevis 2-34
1. prepared by Lactobacillus brevis 2-34 (Lactobacillus brevis 2-34) competent cell
The Lactobacillus brevis 2-34 of glycerol tube preservation is inoculated in MRS fluid nutrient medium by 2% inoculum concentration, 37 DEG C of standing trainings
Support 12h；By the switching of 2% inoculum concentration in the MRS fluid nutrient medium containing 1% glycine, 37 DEG C of stationary cultures are for 24 hours；By 2% inoculation
Amount switching is in the MRS fluid nutrient medium containing 1% glycine, 37 DEG C of stationary cultures to OD600For 0.8-1.0.
It will be centrifuged 10min at 15min, 5000rpm, 4 DEG C of culture solution ice bath, collect thallus；Somatic cells pre-cooling
Washingsolution (WB) is centrifuged 10min at washing 2 times, 6000rpm, 4 DEG C, collect thallus；Somatic cells pre-cooling
Electroporation Buffer (EB) is centrifuged 10min at washing 1 time, 8000rpm, 4 DEG C；Somatic cells 1mL EB weight
It hangs to get competent cell, the packing of 50 μ L/ pipes, -80 DEG C save backup or be immediately available for electrotransformation.
2. plasmid pNZ5913 electrotransformation Lactobacillus brevis 2-34 competent cell
450ng plasmid pNZ5913 is taken, is added in 50 μ L Lactobacillus brevis competent cells, after mixing ice bath 5min, is moved
Enter the electric shock cup (0.2cm spacing) of pre-cooling and shock by electricity, shock by electricity condition 2.4KV, 3.5ms；The MRS liquid of 450 μ L pre-cooling is added immediately
Body culture medium is uniformly mixed with competent cell to the cup that shocks by electricity, moves into the EP pipe of 1.5mL, 37 DEG C of stationary cultures after ice bath 10min
For 2h so that competent cell is recovered, 8000rpm room temperature is centrifuged 10min, carefully topples over supernatant, leaves and takes about 50 μ L bacterium solutions, is coated with
In the MRS plate for containing 30 μ g/ μ L erythromycin, 37 DEG C of stationary culture 4-5d, until there are monoclonal colonies.
3. the identification of positive transformant
The picking transformant extremely MRS fluid nutrient medium containing erythromycin, 37 DEG C of stationary culture 2d are extracted according to bacterial plasmid and are tried
Agent box illustrates to extract plasmid.10 μ L of PCR amplification system is prepared, wherein 0.5 μ L of Plasmid DNA, each 0.1 μ of upstream and downstream primer (F/R)
2 × 5 μ L of L, Primstar Max Premix, 4.3 μ L of sterile water.PCR amplification program are as follows: 98 DEG C of initial denaturation 10min, 98 DEG C of changes
Property 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 10s, 30 circulation, 72 DEG C of extension 10min.
Primer sequence is as shown in table 1.
1 primer sequence of table
| Number | Primer | Sequence (5' → 3') | Sequence number |
| F | pNF | TGAGCGAGGAAGCGGAATA | SEQ ID NO.1 |
| R | pNR | ATAACCTAACTCTCCGTCGC | SEQ ID NO.2 |
As shown in Figure 1, amplified production is identified through 1% agarose gel electrophoresis, size is 1000bp, with expected size
It is consistent, is analyzed after gel extraction through DNA sequencing, amplifiable sequence fits like a glove between F/R primer on sequence and pNZ5913, demonstrate,proves
Bright is positive transformant.
(2) assessment of electrotransformation efficiency
3 μ L (150ng/ μ L) plasmid pNZ5319 converts 50 μ L competent cells, and reaction solution adds 450 μ L MRS culture mediums multiple
Supernatant is removed after Soviet Union 2h, 50 μ L is taken to be coated with, generates 4 bacterium colonies after culture 4-5 days.
According to electrotransformation efficiency calculation formula: electrotransformation efficiency (cfu/ μ g)=generation clump count/bed board DNA total amount,
The electrotransformation efficiency that pNZ5319 converts Lactobacillus brevis 2-34 is 8.9cfu/ μ g, and the method provided according to bibliography, can not
Obtain transformant.
2 plasmid pNZ5319 of embodiment converts Lactobacillus brevis ATCC 367
(1) plasmid pNZ5319 electrotransformation Lactobacillus brevis ATCC 367
According to the competent cell of the method preparation Lactobacillus brevis ATCC 367 in embodiment 1,450ng plasmid is taken
PNZ5913 is added in 50 μ L Lactobacillus brevis ATCC, 367 competent cell, turns according to progress electrotransformation, electricity the step of embodiment 1
It recovers after change, bacterium solution coating, positive transformant is selected and positive transformant identifies that it is table 1 that PCR amplification, which identifies the primer still,
Middle primer sequence.
Positive transformant qualification result is as shown in Fig. 2, amplified production identifies that size is equal through 1% agarose gel electrophoresis
For 1000bp, it is consistent with expected size, is analyzed after gel extraction through DNA sequencing, it can between F/R primer on sequence and pNZ5913
Extension increasing sequence fits like a glove, it was demonstrated that is positive transformant.
(2) assessment of electrotransformation efficiency
3 μ L (150ng/ μ L) plasmid pNZ5319 converts 50 μ L competent cells, and reaction solution adds 450 μ L MRS culture mediums multiple
Supernatant is removed after Soviet Union 2h, 50 μ L is taken to be coated with, generates 7 bacterium colonies after culture 4-5 days.
According to electrotransformation efficiency calculation formula: electrotransformation efficiency (cfu/ μ g)=generation clump count/bed board DNA total amount,
The electrotransformation efficiency that pNZ5319 converts Lactobacillus brevis ATCC 367 is 15.56cfu/ μ g, and the side provided according to bibliography
Method is unable to get transformant.
3 plasmid pNZ5319 of embodiment converts Lactobacillus brevis TMW 1.2112
(1) plasmid pNZ5319 electrotransformation Lactobacillus brevis TMW 1.2112
According to the competent cell of the method preparation Lactobacillus brevis TMW 1.2112 in embodiment 1,450ng plasmid is taken
PNZ5913 is added in 50 μ L Lactobacillus brevis TMW, 1.2112 competent cell, according to progress electrotransformation, electricity the step of embodiment 1
It recovers after conversion, bacterium solution coating, positive transformant is selected and positive transformant identification, PCR amplification identify that the primer is still
Primer sequence in table 1.
Positive transformant qualification result is as shown in figure 3, amplified production identifies that size is equal through 1% agarose gel electrophoresis
For 1000bp, it is consistent with expected size, is analyzed after gel extraction through DNA sequencing, it can between F/R primer on sequence and pNZ5913
Extension increasing sequence fits like a glove, it was demonstrated that is positive transformant.
(2) assessment of electrotransformation efficiency
3 μ L (150ng/ μ L) plasmid pNZ5319 converts 50 μ L competent cells, and reaction solution adds 450 μ L MRS culture mediums multiple
Supernatant is removed after Soviet Union 2h, 50 μ L is taken to be coated with, generates 5 bacterium colonies after culture 4-5 days.
According to electrotransformation efficiency calculation formula: electrotransformation efficiency (cfu/ μ g)=generation clump count/bed board DNA total amount,
The electrotransformation efficiency that pNZ5319 converts Lactobacillus brevis TMW 1.2112 is 11.11cfu/ μ g, and the side provided according to bibliography
Method is unable to get transformant.
The present invention has the electrotransformation method of lactic acid bacteria by improving, and establishes the electrotransformation side of wild type Lactobacillus brevis
Method, the method achieve exogenous dna fragment successful conversions to enter wild type Lactobacillus brevis.It is short using the method for the present invention building wild type
Lactobacillus recombinant bacterial strain, has a good application prospect.
Comparative example 1
1M sucrose, the 3mM MgCl electroporation buffer ingredient middle in embodiment 1 being changed to by 1M sucrose in bibliography2,
Remaining condition is the same as embodiment 1.MgCl2Have the function of removing polysaccharide outside cell wall, the MgCl in resuscitation process2Presence not
Conducive to the recovery of cell wall, and cause bacterium during electric shock breakdown.
Comparative example 2
The opportunity of thallus is collected when prepared by competent cell in embodiment 1 from OD600It is changed to 0.4-0.6 up to 0.8-1.2,
Remaining condition is the same as embodiment 1.Because cell wall structure difference causes different growing stage bacterium to the sensitive journey of electric shock between strain
Degree differs greatly, and most of bacterium is in mid log phase electrotransformation efficiency highest, and part gram-positive bacteria is raw in logarithm
Long latter stage is suitable for electrotransformation.And Lactobacillus brevis 2-34 is insensitive to shocking by electricity in the cell growth early stage stage, electrotransformation efficiency is extremely low.
Comparative example 3
Shock voltage 2.4KV in embodiment 1 when electrotransformation is changed to 2.0KV, remaining condition is the same as embodiment 1.As a result table
Bright, the shock voltage of 2.0KV is lower for Lactobacillus brevis 2-34, and cell membrane reparation speed is relatively fast, leads to exogenous plasmid not
It can timely enter intracellular.
Comparative example 4
Recovery medium MRS culture medium in embodiment 1 is changed to the MRS culture medium of addition 0.3M sucrose, remaining condition is same
Embodiment 1.The result shows that addition 0.3M sucrose is to the conversion of Lactobacillus brevis 2-34 without obvious effect.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of method for transformation of Lactobacillus brevis
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
tgagcgagga agcggaata 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ataacctaac tctccgtcgc 20
Claims (10)
1. a kind of Lactobacillus brevis method for transformation, which is characterized in that the described method includes:
(1) Lactobacillus brevis is activated；
(2) Lactobacillus brevis after activating is cultivated to OD600For 0.8-1.0；
(3) thallus is collected, competent cell is prepared；
(4) it is added in competent cell after exogenous dna fragment through electric shock, electrotransformation Lactobacillus brevis competent cell, voltage setting
For 2.4-2.6KV, the electric shock time is 2-4ms；
(5) competent cell is recovered, and is coated with MRS plate, screens transformant.
2. the method as described in claim 1, which is characterized in that step (1) specifically: Lactobacillus brevis is pressed 1-5% inoculum concentration
It is inoculated in the activation of MRS fluid nutrient medium, 35-38 DEG C of stationary culture 10-14h；Culture solution is transferred in containing by 1-5% inoculum concentration
The MRS fluid nutrient medium of 1% glycine, 35-38 DEG C is continued to activate 20-30h.
3. the method as described in claim 1, which is characterized in that step (2) specifically: will be after activation by 1-5% inoculum concentration
Lactobacillus brevis is inoculated in the MRS fluid nutrient medium containing 1% glycine, 35-38 DEG C of stationary culture to OD600For 0.8-1.0.
4. the method as described in claim 1, which is characterized in that step (3) specifically: the thallus for obtaining step (2) is centrifuged,
Thallus is collected, with Washing Buffer washing thalline cell, thallus is resuspended to get sense with Electroporation Buffer
By state cell.
5. the method as described in claim 1, which is characterized in that step (4) described electric-shocking method is specifically: taking exogenous plasmid
DNA and competent cell mix after in standing 5min on ice, move into electric shock cup, voltage is set as 2.4-2.6KV, and the electric shock time is
2-4ms is transformed into exogenous plasmid dna in competent cell.
6. method as claimed in claim 5, which is characterized in that the ratio of exogenous plasmid dna and competent cell is 400-
500:50 (ng/ μ L).
7. method as claimed in claim 1 or 5, which is characterized in that step (5) specifically includes: immediately to electricity after electric shock
Addition MRS fluid nutrient medium in cup is hit, cell is resuspended to and is transferred to new centrifuge tube, in standing 5-10min on ice, in 35-
Stationary culture 1.5-2h recovers in 38 DEG C of incubators, is centrifuged bacterium solution, abandons part supernatant, thallus is coated on MRS after being resuspended solid
Body culture medium, 35-38 DEG C of stationary culture 4-5d.
8. method as claimed in claim 4, which is characterized in that the Washing Buffer are as follows: 0.5mol/L sucrose,
0.5mmol/L MgCl2,5mmol/L KH2PO4, tune pH are 7.4-7.5,115 DEG C of high pressure sterilization 20min, described
Electroporation Buffer are as follows: 0.5mol/L sucrose, 115 DEG C of high pressure sterilization 20min.
9. the method as described in claim 1, which is characterized in that Lactobacillus brevis includes Lactobacillus brevis 2-34, Lactobacillus brevis ATCC
367 or Lactobacillus brevis TMW 1.2112.
10. application of any method of claim 1-9 in production Lactobacillus brevis intrinsic protein or foreign protein.
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| CN114958896A (en) * | 2021-11-12 | 2022-08-30 | 淮阴师范学院 | Method for improving electrotransformation efficiency of pediococcus acidilactici |
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