CN108251348A - A kind of preparation of thermophilus bacterium competence cell and method for transformation - Google Patents
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Abstract
Preparation and method for transformation the present invention provides a kind of thermophilus bacterium competence cell, by streptococcus thermophilus in LM17 medium cultures to OD600When being 0.3~0.9, with SLM17 culture mediums 1:1 37 DEG C of mixing incubation 1h; 4 DEG C thalline were collected by centrifugation; cell is washed with the buffer solution of precooling; obtain competent cell; after competent cell and pIB184 plasmid mixings; electric field strength is used to shock by electricity for 12.5kV/cm, in LM17MC2 culture mediums 3~8h of recovery, the highest electricity transformation efficiency 1.3 × 10 of streptococcus thermophilus can be obtained6CFU/μg DNA.The present invention method is easy to operate, transformation efficiency is high, genetic modification for streptococcus thermophilus, the molecular mechanism for illustrating phenotypic characteristic provide new technical method.
Description
Technical field
The invention belongs to biotechnologies, are related to a kind of streptococcus thermophilus, specifically a kind of streptococcus thermophilus sense
Preparation and method for transformation by state cell.
Background technology
Report lactic acid bacteria method for transformation is broadly divided into two kinds at present：First, protoplasm body, due to protoplast fragility, makes
It is difficult and very cumbersome that streptococcus thermophilus protoplast must be prepared, while also need to be gone that DNA is promoted to enter with polyethylene glycol.Second is that
Electrotransformation, the method is easy to operate, and transformation efficiency height has been widely used.Electrotransformation is the lactic acid such as current streptococcus thermophilus
Most effective method for transformation in bacterium, and the Efficient Conversion of streptococcus thermophilus is to realize that exogenous genes products are different in streptococcus thermophilus
The premise of source expression.The conversion of streptococcus thermophilus electricity is influenced by many factors, since streptococcus thermophilus is gram-positive bacteria, carefully
Cell wall is thicker, cell is entered to DNA molecular with apparent inhibition, it is to determine electricity conversion to select suitable cell weakening agent
One of successful essential condition.Common attenuator mainly has glycine, threonine, penicillin and lysozyme etc. in lactic acid bacteria.
Other than attenuator, growth conditions, electroporation buffer, electric field strength, plasmid concentration, recovery time and the recovery culture of cell
Base all has an impact electric transformation efficiency.
Streptococcus thermophilus (Streptococcus thermophilus) is a kind of important industrial lactic acid bacteria (Lactic
Acid Bacteria, LAB), unique generally recognized as safe strain is determined as in 93 streptococcuses by USA and EU.As one
The important commercial fermentation agent of kind, is widely used in the production of the fermented dairy products such as Yoghourt and cheese.Streptococcus thermophilus is as prebiotic
Bacterium has Bile salt resistance and gastrointestinal tract adhesiveness, and can synthesize exocellular polysaccharide (Exopolysaccharides, EPS).It is thermophilic
Hot streptococcus EPS is applied to as tackifier, stabilizer, emulsifier and gelling agent in food, can significantly improve the mouth of dairy products
Sense and rheological behavior；In addition, EPS has potential antitumor, norcholesterol, adjusts immune system and the various actives such as anti-oxidant
Function.In order to illustrate the molecular mechanism of streptococcus thermophilus phenotypic characteristic at the genetic level, streptococcus thermophilus must be carried out
Genetic manipulation, therefore it is most important to establish efficient streptococcus thermophilus method for transformation.
PIB184 plasmids are Escherichia coli-lactic acid bacteria shuttle plasmid, are a high copy expression carriers, contain P23Strong composition
The elements such as type promoter and multiple cloning sites, size~6kb；Working concentration in Escherichia coli is 200~300 μ g/mL,
5~20 μ g/mL of working concentration in lactic acid bacteria.PIB184 plasmids are built by scholars such as kansas, U.S.A university Indranil
(INDRANIL B,JYOTI K.J,NICHOLAS F.Shuttle expression plasmids for genetic
studies in Streptococcus mutans.Microbiology,2008,154:2275-2282), extensively by the whole world
Colleague is used.Such as the research such as CHATTORAJ is dense using the work of pIB184 plasmids in Streptococcus mutans
It spends for 5 μ g/mL (CHATTORAJ P, MOHAPATRA S.S, BISWAS I etc.Regulation of transcription
by SMU.1349,a TetR family regulator,in Streptococcus mutans.Journal of
Bacteriology,2011,193:6605-6613).Liu Zhenmin etc. is using the working concentration of pIB184 in Lactobacillus casei
(Li Nan, apple of going on a spring outing, Liu Zhen people .NADH Oxidase Expressions are to the influence of Lactobacillus casei LC2W yield of extracellular polysaccharide by 10 μ g/mL
Food industry science and technology, 2016,6:182-186).
Invention content
For above-mentioned technical problem of the prior art, the present invention provides a kind of systems of thermophilus bacterium competence cell
Standby and method for transformation, the preparation of this thermophilus bacterium competence cell and method for transformation will solve thermophilic in the prior art
The technical issues of method transformation efficiency of hot streptococcus electricity conversion is not high.
Preparation and method for transformation the present invention provides a kind of thermophilus bacterium competence cell, include the following steps：
1) by streptococcus thermophilus in LM17 medium cultures to OD600When being 0.3~0.9, with SLM17 culture mediums according to body
Product ratio 1:1 is blended in 37 DEG C of 1~2h of incubation, and 2~6 DEG C thalline were collected by centrifugation, washs cell with the buffer solution of 0~4 DEG C of precooling, obtains
To competent cell；
2) it is 100 μ L according to material ratio with pIB184 plasmids by the competent cell that step 1) obtains：50~200ng ratios
After mixing, electric field strength is used to shock by electricity for 12.5~15kV/cm, in LM17MC2 culture mediums 3~8h of recovery, obtain high electricity conversion
The streptococcus thermophilus of efficiency.
Further, by streptococcus thermophilus in LM17 medium cultures to OD600It is 0.3.
Further, the precooling buffer solution is the solution containing 0.4M sorbierites and 5mM dipotassium hydrogen phosphates.
Further, electric field strength is used to shock by electricity for 12.5kV/cm.
Further, in LM17MC2 culture medium recoveries 6h.
Further, in the LM17 culture mediums, every liter of culture medium contains 5g tryptones, 5g soy peptones,
2.5g yeast extract powders, 5g beef extracts, 20g lactose, 19g β-five hydrate of phosphoglycerol disodium, 0.58g epsom salts,
0.5g vitamin Cs.
Further, the SLM17 culture mediums are the LM17 culture mediums containing glycine, and in the medium, described is sweet
The mass percent concentration of propylhomoserin is 5%.
Further, the LM17MC2 recovery medias is contain sorbierite, CaCl2、MgCl2LM17 culture mediums,
In the medium, a concentration of 0.2M containing sorbierite, the CaCl2A concentration of 2mM, the MgCl2It is dense
It spends for 20mM.
Preparation and method for transformation the present invention provides a kind of thermophilus bacterium competence cell, method using the present invention
It is converted, the highest electricity transformation efficiency 1.3 × 10 of streptococcus thermophilus can be obtained6CFU/μg DNA。
The present invention is compared with prior art, and technological progress is significant.Method of the invention is easy to operate, transformation efficiency
Height, genetic modification for streptococcus thermophilus, the molecular mechanism for illustrating phenotypic characteristic provide new technical method.
Description of the drawings：
Fig. 1 is verified for transformant plasmid extraction and PCR；(A)M：DL 10000marker；1~7：Transformant extracts plasmid
Xho I single endonuclease digestion electrophoresis results.(B)M：DL 2000marker；1~7：Erythromycin (Em) resistant gene PCR results.
Fig. 2 is influence of the different electric field strengths to AR479 electricity transformation efficiencies.
Fig. 3 is influence of the different recovery times to AR479 electricity transformation efficiencies.
Fig. 4 is the influence of different glycine concentrations and incubation time to AR479 electricity transformation efficiencies.
Fig. 5 is influence of the different recovery mediums to AR479 electricity transformation efficiencies；(A)1：LM17MC1；2：LM17MC2；3：
LM17MC3；4：LM17MC4.
Specific embodiment
The specific DNA sequence dna information of bacterial strain, plasmid and primer used in all embodiments is shown in Tables 1 and 2.All embodiments
In involved main agents such as Taq archaeal dna polymerases, restriction enzyme XhoI, DNA Marker DL2000 and DNA
Marker DL10000 are purchased from TaKaRa companies；Small amount plasmid extraction kit, erythromycin, chloramphenicol and ampicillin
Sangon Biotech (Shanghai) Co., Ltd. is purchased from other chemicals.
The culture medium used in all embodiments is as follows：
LM17 culture mediums：For the activation, culture and count plate of AR479.It is formulated as (g/L)：Tryptone 5, soybean
Peptone 5, yeast extract powder 2.5, beef extract 5, lactose 20, five hydrate 19 of β-phosphoglycerol disodium, epsom salt
0.58, vitamin C 0.5.115 DEG C of sterilizing 20min.Solid medium separately adds agar powder 20.
SLM17 culture mediums：LM17 culture mediums containing 5% glycine.
LM17MC1 recovery medias：LM17 culture mediums containing 0.2M sorbierites.
LM17MC2 recovery medias：Contain 0.2M sorbierites, 2mM CaCl2、20mM MgCl2LM17 culture mediums.
LM17MC3 recovery medias：LM17 culture mediums containing 0.4M sucrose.
LM17MC4 recovery medias：Contain 0.4M sucrose, 2mM CaCl2、20mM MgCl2LM17 culture mediums.
LB culture mediums：For the culture of E.coli.It is formulated as (g/L)：Tryptone 10, dusty yeast 5, sodium chloride 10.121
DEG C sterilizing 15min.Solid medium separately adds agar powder 20.
The present invention is further explained with reference to embodiments.For specific method or material used in embodiment, sheet
Field technology personnel can carry out conventional replacement according to existing technology and select on the basis of the technology of the present invention thinking, and
It is not limited only to the specific record of the embodiment of the present invention.
1 bacterial strain of table and plasmid
The primer used in 2 researchs of table
Experimental method used in following embodiments is conventional method unless otherwise specified；Used material, examination
Agent etc., is commercially available unless otherwise specified.
Embodiment 1：The foundation of streptococcus thermophilus AR479 competence preparation methods
According to the research of lactic acid bacteria electricity method for transformation reported in the literature, 5 kinds of common competence systems below have been first attempted to
Preparation Method：
Method one：- 80 DEG C of glycerol stocks bacterium AR479 scribing line are activated, LM17 Liquid Cultures are stayed overnight, the inoculation of 1% inoculum concentration
To the LM17 for containing threonine (100mM), 37 DEG C of cultures to OD6000.6~0.8,4 DEG C of 5000x g centrifuge 10min.It is slow with washing
Fliud flushing (0.5M sucrose, 10% glycerine) washes twice, then heavy with buffer solution (10% glycerine and 30%PGE2000 solution) is resuspended
Outstanding, often 100 μ L of pipe, -80 DEG C of preservation, electricity conversion are spare.
Method two：LM17 Liquid Cultures are stayed overnight, and 1% inoculum concentration is seeded to the LM17 of lysozyme (10mg/mL), 37 DEG C of trainings
It supports to OD6000.6~0.8,4 DEG C of 5000x g centrifuge 10min.It is washed with washing buffer (0.5M sucrose and 10% glycerite)
It washs twice, then be resuspended with buffer solution (10% glycerine and 30%PGE2000 solution) is resuspended, often 100 μ L of pipe, -80 DEG C of preservation, electricity turns
Change spare.
Method three：LM17 Liquid Cultures are stayed overnight, and 1% inoculum concentration is seeded to the LM17 containing threonine (20mM), 37 DEG C of cultures
To OD6000.2~0.5,4 DEG C of 5000x g centrifuge 15min.With buffer solution (7mM HEPES and 1mM MgCl2PH value of solution 6.0)
It washes twice, is then resuspended, often 100 μ L of pipe, -80 DEG C of preservation, electricity converts spare.
Method four：LM17 Liquid Cultures are stayed overnight, and 1% inoculum concentration is seeded to containing glycine (0.5%, 1.0%, 1.5% and
2.0%) LM17,37 DEG C of cultures to OD6000.2~0.4,4 DEG C of 5000x g centrifuge 15min.With buffer solution (7mM HEPES,
272mM sucrose and 1mM EDTA pH value of solution 6.5) it washes twice, it is then resuspended, often 100 μ L of pipe, -80 DEG C of preservation, electricity conversion is standby
With.
Method five：LM17 Liquid Cultures are stayed overnight, and 3% is inoculated in the LM17 culture mediums of 42 DEG C of preheatings, surveys OD600About 0.3,
1:1 ratio is inoculated in 0.1% glycine LM17 culture mediums, cultivates 2h, and 4 DEG C of 4000rpm centrifuge 10min.With buffer solution (Tris
6.057g, glycine 28.8g, SDS 10g adjust pH 8.3 and are settled to 1000mL) it washes twice, it is then resuspended, 100 μ L are every
Pipe, -80 DEG C of preservation, electricity conversion are spare.
The streptococcus thermophilus AR479 competence prepared using above-mentioned 5 kinds common competence preparation methods, respectively by 7 kinds of breasts
Sour bacteria plasmid electricity is converted into above-mentioned competence, but the unconverted success (table 3) of exogenous plasmid.
Therefore, the present invention devises a kind of new thermophilus bacterium competence preparation method (abbreviation method six), specific to walk
It is rapid as follows：
Picking single bacterium colony LM17 Liquid Cultures are stayed overnight, and 3% inoculum concentration is seeded to the 37 DEG C of cultures of LM17 culture mediums to OD600
0.3~0.5, with SLM17 culture mediums 1:1 37 DEG C of mixing is incubated 1h, 4 DEG C of 5000x g, and centrifugation 5min collects thalline.With 0~4 DEG C
Twice, competence is divided for electroporation buffer (0.4M sorbierites and 5mM dipotassium hydrogen phosphate solutions, pH 4.5) the washing cell of precooling
It fills 100 μ L often to manage, -80 DEG C of preservation, electricity conversion is spare.
Using method six prepare streptococcus thermophilus AR479 competence, respectively by 7 kinds of plasmid electricity described in following table convert to
(according to material ratio it is 100 μ L by competent cell and pIB184 plasmids in competence：100ng ratios mixing), enable AR479
Success absorbs exogenous plasmid, enables in two kinds of lactic acid bacteria plasmid pIB184 and pNZ44 successful conversions to AR479.
The different plasmid electricity transformation efficiencies of table 3
Nd indicates no transformant.
Whether it is positive to obtain cloning after the above-mentioned conversion of verification, the present invention selects 7 pIB184 plasmids at random and successfully turns
The positive colony of change uses Xho I single endonuclease digestions after extracting plasmid, and agarose gel electrophoresis analysis has list in 7000bp positions or so
One band, size are correct (Figure 1A).Erythromycin resistance gene fragment primer (table 2) is selected to carry out bacterium colony PCR verifications, there is band
And (Figure 1B) in the same size with target fragment.Experiment shows in pIB184 plasmids successful conversion to streptococcus thermophilus AR479.
Embodiment 2：The optimization of streptococcus thermophilus AR479 electricity conversion conditions
In electric conversion process, electric field strength is key factor.Electroporation is using instantaneous pressure electric shock cell membrane, is allowed to shape
Into hole, allow DNA molecular that can enter cell.It is too low or too high for voltage all to have exogenous DNA molecule during electric shock
Effect enters cell.However, in certain electric field strength range, electric field strength is stronger, and the hole generated on cell membrane is more, leads to
Permeability is better, and exogenous DNA molecule is easier to enter cell.AR479 electricity transformation efficiencies improve (Fig. 2) with the increase of electric field strength,
When electric field strength reaches 12.5kV/cm, electric transformation efficiency reaches highest, is 6 × 105CFU/μg DNA.With electric field strength
Further increase, transformation efficiency is declined.
Cell after high-voltage electric shock and the glycine of high concentration are incubated, perforated, and selects hypertonic culture medium pair by cell membrane
The recovery of cell is of crucial importance.Fig. 3 shows that in a certain range the recovery time is longer, and electric transformation efficiency is higher, and in 6h, electricity turns
Change efficiency and reach maximum (6 × 105CFU/μg DNA).When recovery is more than 6h, electric transformation efficiency declines.The recovery time is too short,
The perforation on cell membrane may be made to have little time to be closed or repair, cause transformation efficiency relatively low；And the recovery time is too long, due to lacking
The screening pressure of corresponding antibiotic may cause the loss of plasmid, also result in that clone is on the low side, and transformation efficiency is relatively low.
Since the cell wall of lactic acid bacteria is thicker, the absorption of exogenous plasmid is influenced, therefore is added in the medium suitably
Chemical substance (such as：Lysozyme, penicillin, threonine, glycine etc.) influence the synthesis of cell wall, enable exogenous plasmid
Into cell.Present invention selection glycine processing cell, adds the glycine of various concentration and is determining sweet ammonia in the medium
Change the incubation time of glycine and cell under acid concentration to improve electric transformation efficiency.By in Fig. 4 A it is found that in glycine concentration
It is 5%, when being incubated 1h with glycine, electric transformation efficiency reaches highest 1 × 106CFU/ μ g DNA, with carrying for glycine concentration
Height, transformation efficiency are gradually reduced.According to the best glycine concentration 5% of acquisition, by glycine and cell incubation different time, knot
When fruit shows incubation time as 1h, electric transformation efficiency highest extends time transformation efficiency and declines instead (Fig. 4 B).
Mortality is easy to after cell electric shock, the survival rate of cell can be significantly improved by recovering in hypertonic environment.This
Invention is using 4 kinds of hypertonic culture mediums come the AR479 cells after electricity conversion of recovering, the results showed that electricity turns in LM17MC2 culture mediums
Change efficiency and be up to 1.1 × 106CFU/ μ g DNA (Fig. 5 A).4 kinds of culture mediums are tied using different sugar protection cells from experiment
Fruit illustrates that sorbierite is more advantageous to AR479 cell membranes it is found that the transformation efficiency of the sorbierite of same concentrations is apparently higher than sucrose
Restore.The present invention it is also noted that LM17MC2 recovery medias than LM17MC1 recovery media transformation efficiency higher, it may be possible to
Due to CaCl2And MgCl2It can promote the absorption of exogenous DNA, obtain more transformants.To further improve efficiency, according to upper
On the basis of stating best recovery medium LM17MC2, sorbitol concentration is advanced optimized, by Fig. 5 B it is found that containing 0.2M sorbs
Transformation efficiency highest in the LM17MC2 culture mediums of alcohol, reaches 1.3 × 106CFU/ μ g DNA, when sorbitol concentration is too high or too low
All it is unfavorable for the recovery of cell.
Electricity conversion at present is that foreign gene imports streptococcus thermophilus most efficient method, and easy to operate, but it is converted
Efficiency is generally relatively low, constrains streptococcus thermophilus molecular biology research, therefore it is particularly critical to establish efficient electric method for transformation.
The present embodiment establishes new streptococcus thermophilus electricity method for transformation, successfully realizes that exogenous plasmid is converted to streptococcus thermophilus AR479
In.It is carried out for the electricity conversion key factor such as electric field strength, glycine concentration and incubation time, recovery medium and recovery time
Optimization obtains the highest electricity transformation efficiency 1.3 × 10 of report streptococcus thermophilus at present6CFU/μg DNA.To streptococcus thermophilus electricity
The transformation efficiency of Study on Transformation, Buckley etc. are 102~104CFU/μg DNA(BUCKLEY N D,VADEBONCOEUR C,
LEBLANC D J,et al.An effective strategy,applicable to Streptococcus
salivarius and related bacteria,to enhance or confer electroporation
competence.Applied and Environmental Microbiology,1999,65(9):3800-3804.),
The electricity conversion peak efficiency such as George is only 102~103CFU/μg DNA(SOMKUTI G A,STEINBERG D H.Genetic
transformation of Streptococcus thermophilus by electroporation.Biochimie,
1988,70(4):579-585.), the transformation efficiencies such as Marciset are up to 8 × 105CFU/μg DNA(MARCISET O,
MOLLET B.Multifactorial experimental design for optimizing transformation:
Electroporation of Streptococcus thermophilus.Biotechnology and
Bioengineering,1994,43(6):490-496.), the electric transformation efficiency of these methods is below the conversion effect of this research
Rate.
Above example is only the basic explanation under present inventive concept, is not limited the invention.And according to the present invention
Any equivalent transformation for being made of technical solution, all belong to the scope of protection of the present invention.
Sequence table
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Claims (8)
1. preparation and the method for transformation of a kind of thermophilus bacterium competence cell, it is characterised in that include the following steps：
1) by streptococcus thermophilus in LM17 medium cultures to OD600When being 0.3~0.9, with SLM17 culture mediums according to volume ratio
1:1 is blended in 37 DEG C of 1~2h of incubation, and 2~6 DEG C thalline were collected by centrifugation, washs cell with the buffer solution of 0~4 DEG C of precooling, is felt
By state cell；
2) it is 100 μ L according to material ratio with pIB184 plasmids by the competent cell that step 1) obtains：50~200ng ratio mixings
Afterwards, electric field strength is used to shock by electricity for 12.5~15kV/cm, in LM17MC2 culture mediums 3~8h of recovery, obtains high electric transformation efficiency
Streptococcus thermophilus.
2. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
By streptococcus thermophilus in LM17 medium cultures to OD600It is 0.3.
3. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
The precooling buffer solution is the solution containing 0.4M sorbierites and 5mM dipotassium hydrogen phosphates.
4. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
Electric field strength is used to shock by electricity for 12.5kV/cm.
5. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
In LM17MC2 culture medium recoveries 6h.
6. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
In the LM17 culture mediums, every liter of culture medium contains 5g tryptones, 5g soy peptones, 2.5g yeast extract powders, 5g
Beef extract, 20g lactose, 19g β-five hydrate of phosphoglycerol disodium, 0.58g epsom salts, 0.5g vitamin Cs.
7. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
The SLM17 culture mediums are the LM17 culture mediums containing glycine, in LM17 culture mediums, the quality percentage of the glycine
Specific concentration is 5%.
8. preparation and the method for transformation of a kind of thermophilus bacterium competence cell according to claim 1, it is characterised in that:
The LM17MC2 recovery medias is contain sorbierite, CaCl2、MgCl2LM17 culture mediums, in LM17MC2 renewal cultivations
In base, a concentration of 0.2M containing sorbierite, the CaCl2A concentration of 2mM, the MgCl2It is a concentration of
20mM。
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| CN113151022A (en) * | 2021-05-28 | 2021-07-23 | 武汉华美生物工程有限公司 | Preparation method and transformation method of pichia pastoris efficient transformation competent cell |
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| CN113151022A (en) * | 2021-05-28 | 2021-07-23 | 武汉华美生物工程有限公司 | Preparation method and transformation method of pichia pastoris efficient transformation competent cell |
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