CN105567725A - Electricity transformation method for lactobacillus casei - Google Patents
Electricity transformation method for lactobacillus casei Download PDFInfo
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- CN105567725A CN105567725A CN201610053312.8A CN201610053312A CN105567725A CN 105567725 A CN105567725 A CN 105567725A CN 201610053312 A CN201610053312 A CN 201610053312A CN 105567725 A CN105567725 A CN 105567725A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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Abstract
The invention relates to the field of molecular biology, and discloses an electricity transformation method for lactobacillus casei. The electricity transformation method includes the specific methods and the specific steps of electricity transformation of the lactobacillus casei. The problems that traditional electricity transformation is poor in repeatability and low in conversion rate are solved. The reasonably-optimized electricity transformation method for the lactobacillus casei can be applied to DNA expression of multiple different external sources, and efficient expression of biological medicine genes is potentially promoted.
Description
Technical field
The present invention relates to biology field, particularly a kind of lactobacterium casei electricity method for transformation.
Background technology
The importance that Bacterium lacticum has great economic implications at agricultural, field of food and generally acknowledges animal and human's body health, more and more causes the concern of people.For centuries, Bacterium lacticum has been widely used in food and beverage processing industry as important probiotic bacterium, and its biological characteristics is bar-shaped, anaerobism or micro-aerobic, belongs to easily raw bacterium.20% of the total fermentation based food in the whole world is accounted for using Bacterium lacticum as the economic worth that zymogenic foodstuffs industry creates.Simultaneously Bacterium lacticum is also widely used in intestines, the processing of meat-based food and fresh-keeping.Bacterium lacticum by rebuilding intestinal microflora balance, thus is conducive to the mankind and some animal host recovers and improves state of health.
Due to Bacterium lacticum industrial use and significance medically widely, focus is become to Bacterium lacticum molecule genetics research, particularly utilizing genetic engineering technique, is that carrier foreign gene-carrying (as antigen, enzyme, little peptide molecule etc.) directly produces exogenous protein in humans and animals body with Bacterium lacticum.The prerequisite of the genetic manipulation of Bacterium lacticum sets up convenient and reliable method for transformation.The method for transformation of current Bacterium lacticum has protoplast transformation and electroporated method.Protoplast transformation occurs the earliest, but has loaded down with trivial details, consuming time and unstable shortcoming and abandoned, and a kind of lactobacterium casei electricity method for transformation instead of protoplast transformation, but still very imperfect, especially repeated bad, low conversion rate.
Summary of the invention
The object of the present invention is to provide a kind of lactobacterium casei electricity method for transformation, transform repeated bad to overcome traditional electrical, the problem of low conversion rate.
For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the invention discloses a kind of lactobacterium casei electricity method for transformation, method includes lactobacterium casei cultivation, lactobacterium casei electricity is cultivated after transforming and transforming again, and concrete steps are:
Lactobacterium casei is cultivated:
A. take out a species L. casei adding glycerine and preserve from cryogenic freezing refrigerator, MRS solid medium is rule, 37 DEG C of anaerobism are inverted and are cultivated;
B. connect in bacterium colony that collarium picking grown on MRS to test tube in MRS liquid nutrient medium with aseptic, 37 DEG C of static gas wave refrigerator spend the night;
C. the bacterium liquid 1mL that the day before yesterday has grown by second day is transferred in 50mLMRS liquid, when 37 DEG C of static growth to bacterium liquid 600nm place ultraviolet absorption values are 0.5-0.6, prepare lactobacterium casei electricity and transforms;
Lactobacterium casei electricity transforms:
D. lactobacterium casei is moved in 50mL centrifuge tube, be put in ice chest and leave standstill after 20 minutes, be put into and be pre-chilled to 5000r/min in 4 DEG C of whizzers in advance, 5 minutes, discard supernatant, leave precipitation; To precipitation in move into 40mLEPWB solution, precipitation is suspended in EPWB solution, is put into 5000r/min in the whizzer being pre-chilled to 4 DEG C in advance, 5 minutes, discard aqueous phase, leave thalline, this step repeats 3 times, with 40mLEPB solution washing thalline once, abandon supernatant;
E. thalline is resuspended with the EPB solution of 400 μ L, and packing 500-300 μ L/ props up (matching while using), turns for electricity;
F. in the lactobacterium casei competent cell that plasmid DNA 1 μ g prepares before joining, mix gently, ice bath 10 minutes;
G. 2mm plasmid and lactobacterium casei competence being proceeded to precooling in advance shocks by electricity in cup, ice bath 10 minutes;
H. with filter paper, electricity is transformed cup surface to dry, put into electroporation, regulating voltage 2.3kV, resistance 200 Ω, electric capacity 25 μ F, the burst length is about 4-5ms;
I. add fast in electric shock cup 900 μ L containing the MRS of 0.5% glycine, move on to after mixing with pipettor pressure-vaccum lightly in an aseptic Eppendorf pipe, be put into be adjusted to 37 DEG C in advance 80-100rpm/min shaking table in hatch 1.5-3 hour;
Cultivate again after conversion:
J. the mixed solution after hatching is centrifugal, leaves 100 μ L supernatants, Eddy diffusion; Then coat on the MRS agar plate containing selective pressure, 37 DEG C of anaerobics are inverted and are cultivated 24-36 hour.
Wherein, MRS substratum is mupirocin lithium salts modified MRS culture medium, and EPWB solution is: NaH
2pO
40.6mmol/L and MgCl
20.1mmol/L configuration forms, and EPB solution is that EPWB solution basis adds 0.3mol/L sucrose, regulates pH to be 7.4, and sterilizing 4 DEG C preservation.
The present invention has following beneficial effect:
1. the invention discloses concrete grammar and the step of the conversion of lactobacterium casei electricity, overcome traditional electrical and transform repeated bad, the problem of low conversion rate.2., through the lactobacterium casei electricity method for transformation of reasonably optimizing, multiple different foreign DNA can be applied to and express, the potential high expression having promoted bio-pharmaceutical gene.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.
Embodiment 1
The invention discloses a kind of lactobacterium casei electricity method for transformation, method includes lactobacterium casei cultivation, lactobacterium casei electricity is cultivated after transforming and transforming again, and concrete steps are:
Lactobacterium casei is cultivated:
A. take out a species L. casei adding glycerine and preserve from cryogenic freezing refrigerator, MRS solid medium is rule, 37 DEG C of anaerobism are inverted and are spent the night;
B. connect bacterium colony that collarium picking grown on MRS in the MRS liquid nutrient medium of 3-5mL with aseptic, 37 DEG C of static gas wave refrigerator spend the night;
C. the bacterium liquid 1mL that the day before yesterday has grown by second day is transferred in the MRS liquid of 50mL, when 37 DEG C of static growth to bacterium liquid 600nm place ultraviolet absorption values are 0.5-0.6, prepare lactobacterium casei electricity and transforms;
Lactobacterium casei electricity transforms:
D. lactobacterium casei is moved in 50mL centrifuge tube, be put in ice chest and leave standstill after 20 minutes, be put into and be pre-chilled to 5000r/min in 4 DEG C of whizzers in advance, 5 minutes, discard supernatant, leave precipitation; To precipitation in move into 40mLEPWB solution, precipitation is suspended in EPWB solution, is put into 5000r/min in the whizzer being pre-chilled to 4 DEG C in advance, 5 minutes, discard aqueous phase, leave thalline, this step repeats 3 times, with 40mLEPB solution washing thalline once, abandon supernatant;
E. thalline is resuspended with the EPB solution of 400 μ L, and packing 50-300 μ L/ props up (matching while using), turns for electricity;
F. in the lactobacterium casei competent cell that plasmid DNA 1 μ g prepares before joining, mix gently, ice bath 10 minutes;
G. 2mm plasmid and lactobacterium casei competence being proceeded to precooling in advance shocks by electricity in cup, ice bath 10 minutes,
H. with filter paper, electricity is transformed cup surface to dry, put into electroporation, regulating voltage 2.3kV, resistance 200 Ω, electric capacity 25 μ F, the burst length is about 4-5ms;
I. add fast in electric shock cup 900 μ L containing the MRS of 0.5% glycine, move on to after mixing with pipettor pressure-vaccum lightly in an aseptic Eppendorf pipe, be put into be adjusted to 37 DEG C in advance 80-100rpm/min shaking table in hatch 1.5-3 hour;
Cultivate again after conversion:
J. the mixed solution after hatching is centrifugal, leaves 100 μ L supernatants, Eddy diffusion bacterium, then coats on the MRS agar plate containing selective pressure, and 37 DEG C of anaerobics are inverted and are cultivated 24-36 hour; Wherein, MRS substratum is mupirocin lithium salts modified MRS culture medium, and EPWB solution is: NaH
2pO
40.6mmol/L and MgCl
20.1mmol/L configuration forms, and EPB solution is that EPWB solution basis adds 0.3mol/L sucrose, regulates pH to be 7.4, and sterilizing 4 DEG C preservation.
Principle of work:
Utilize high-voltage pulse current can make cell walls that the reversibility perforation of moment occurs, foreign DNA is transferred in cell through cytolemma, after certain condition recovery, the aperture on cytolemma will be closed again, prevent intracellular organic matter from outflowing, but because lactobacillus is in gram-positive microorganism, cell wall thickness, foreign DNA is difficult to enter cell, causes electric transfer efficient low, repeatability is not high, limits the research to its gene level.After overtesting is groped repeatedly, establish the milk-acid bacteria after electric shock to need in the MRS nutrient solution of supplementary 0.3mol/L sucrose, 37 DEG C of static gas wave refrigerator 1.5-3 hour, then the committed step of spread plate is set up, the effective situation reducing the rear cell of electric shock and break in MRS substratum.
Embodiment 2
Experiment purpose and method: in order to method of contrast optimizes front and back Bacterium lacticum electricity transformation efficiency change, as described in Example 1, the working method before optimization is as follows for the working method after wherein optimizing:
By in Bacterium lacticum list bacterium colony access MRS nutrient solution, after 37 DEG C of quiescent culture spend the night, with in 2% inoculum size access 20mlMRS nutrient solution, 37 DEG C of quiescent culture are about about 4h, to nutrient solution liquid 600nm place ultraviolet absorption value 0.15-0.18, collected by centrifugation thalline, with isopyknic ice-cold electroporation buffer washed cell 2 times.Culture is suspended in 200 μ l electroporation buffer.
Get after 1 μ l plasmid DNA mixes with the ice-cold cell suspension of 40 μ l, move into the mid-5min on ice of electric shock cup, voltage 1175kV is set, resistance 100 Ω, electric capacity 25 μ F, then discharge electricimpulse, shock by electricity the complete MRS nutrient solution adding 400 μ l in cup, then suck in Eppendorf tube, 37 DEG C of static gas wave refrigerator 2 ~ 3h, get on MRS flat board that 100 μ l are coated on containing paraxin, cultivate 24-36h, concrete operations are with reference to relevant criterion operational manuals such as " microbial techniques ".
Experimental result:
Front and back transformation efficiency synopsis optimized by table 1
Note: transformation efficiency is that per unit mass DNA molecular obtains conversion subnumber, and unit is for transforming the every micrograms of DNA molecule of subnumber.
As shown in table 1, the average conversion before and after being optimized by contrast after many experiments can be found, the transformation efficiency after optimization comparatively optimize before transformation efficiency have the raising of more than 50 times, overcome traditional electrical method for transformation repeatability bad, the problem of low conversion rate.
The above; be only the present invention's preferably embodiment, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.
Claims (2)
1. a lactobacterium casei electricity method for transformation, is characterized in that, described method includes lactobacterium casei cultivation, lactobacterium casei electricity is cultivated after transforming and transforming again, and concrete steps are:
Lactobacterium casei is cultivated:
A. take out a species L. casei adding glycerine and preserve from cryogenic freezing refrigerator, MRS solid medium is rule, 37 DEG C of anaerobism are inverted and are cultivated;
B. connect in bacterium colony that collarium picking grown on MRS to test tube in MRS liquid nutrient medium with aseptic, 37 DEG C of static gas wave refrigerator spend the night;
C. the bacterium liquid 1mL that the day before yesterday has grown by second day is transferred in 50mLMRS liquid, when 37 DEG C of static growth to bacterium liquid 600nm place ultraviolet absorption values are 0.5-0.6, prepare lactobacterium casei electricity and transforms;
Lactobacterium casei electricity transforms:
D. lactobacterium casei is moved in 50mL centrifuge tube, be put in ice chest and leave standstill after 20 minutes, be put into and be pre-chilled to 5000r/min in 4 DEG C of whizzers in advance, 5 minutes, discard supernatant, leave precipitation; To precipitation in move into 40mLEPWB solution, precipitation is suspended in EPWB solution, is put into 5000r/min in the whizzer being pre-chilled to 4 DEG C in advance, 5 minutes, discard aqueous phase, leave thalline, this step repeats 3 times, with 40mLEPB solution washing thalline once, abandon supernatant;
E. thalline is resuspended with the EPB solution of 400 μ L, and packing 500-300 μ L/ props up (matching while using), turns for electricity;
F. in the lactobacterium casei competent cell that plasmid DNA 1 μ g prepares before joining, mix gently, ice bath 10 minutes;
G. 2mm plasmid and lactobacterium casei competence being proceeded to precooling in advance shocks by electricity in cup, ice bath 10 minutes;
H. with filter paper, electricity is transformed cup surface to dry, put into electroporation, regulating voltage 2.3kV, resistance 200 Ω, electric capacity 25 μ F, the burst length is about 4-5ms;
I. add fast in electric shock cup 900 μ L containing the MRS of 0.5% glycine, move on to after mixing with pipettor pressure-vaccum lightly in an aseptic Eppendorf pipe, be put into be adjusted to 37 DEG C in advance 80-100rpm/min shaking table in hatch 1.5-3 hour;
Cultivate again after conversion:
J. the mixed solution after hatching is centrifugal, leaves 100 μ L supernatants, Eddy diffusion; Then coat on the MRS agar plate containing selective pressure, 37 DEG C of anaerobics are inverted and are cultivated 24-36 hour.
2. a kind of lactobacterium casei electricity method for transformation as claimed in claim 1, is characterized in that: described MRS substratum is mupirocin lithium salts modified MRS culture medium, and described EPWB solution is: NaH
2pO
40.6mmol/L and MgCl
20.1mmol/L configuration forms, and described EPB solution is that EPWB solution basis adds 0.3mol/L sucrose, regulates pH to be 7.4, and sterilizing 4 DEG C preservation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365802A (en) * | 2017-06-12 | 2017-11-21 | 苏州系统医学研究所 | A kind of electric method for transformation on Listeria |
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| CN103266127B (en) * | 2013-05-03 | 2014-10-01 | 安徽工程大学 | Method for converting bacillus subtilis by electric shock |
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2016
- 2016-01-27 CN CN201610053312.8A patent/CN105567725A/en active Pending
Patent Citations (6)
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| CN101225381A (en) * | 2007-12-30 | 2008-07-23 | 侯喜林 | Lactic acid bacillus transformation method |
| CN101525589A (en) * | 2009-01-14 | 2009-09-09 | 东北农业大学 | Recombinant lactobacillus casei for expressing infectious pancreas necrosis virus (IPNV) VP3 protein and preparation method |
| CN101948857B (en) * | 2010-08-16 | 2013-02-13 | 中国农业大学 | Recombinant lactobacillus casei engineering strain and preparation method thereof |
| CN103820484A (en) * | 2012-11-16 | 2014-05-28 | 天津科技大学 | Electroporation genetic transformation method of lactobacillus plantarum |
| CN103266127B (en) * | 2013-05-03 | 2014-10-01 | 安徽工程大学 | Method for converting bacillus subtilis by electric shock |
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| ANDREA EISEN 等: "Effect of culture media on Lactobacillus Hydrophobicity and electrophoretic mobility", 《MICROB ECOL》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365802A (en) * | 2017-06-12 | 2017-11-21 | 苏州系统医学研究所 | A kind of electric method for transformation on Listeria |
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Inventor after: Hou Xilin Inventor after: Yu Liyun Inventor before: Yu Liyun Inventor before: Hou Xilin |
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Application publication date: 20160511 |