CN103160493A - Method of rapidly eliminating antibiotic-resistant plasmids in E. coli - Google Patents
Method of rapidly eliminating antibiotic-resistant plasmids in E. coli Download PDFInfo
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- CN103160493A CN103160493A CN 201110422779 CN201110422779A CN103160493A CN 103160493 A CN103160493 A CN 103160493A CN 201110422779 CN201110422779 CN 201110422779 CN 201110422779 A CN201110422779 A CN 201110422779A CN 103160493 A CN103160493 A CN 103160493A
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Abstract
A method of rapidly eliminating antibiotic-resistant plasmids in E. coli is mainly used for rapidly and effectively eliminating antibiotic-resistant plasmids from the E. coli. A method of electric shock for the E. coli is used to eliminate the antibiotic-resistant plasmids in the E. coli, and a special electric shock buffer is used to promote elimination of antibiotic-resistant plasmids, thereby improving elimination rate of the plasmids. When cells are in an electric field, a voltage reaches a critical value, localized cell membranes are punctured to form instantaneous pores, wherein pore size is suitable for discharging of the plasmids from the cells. After the electric shock, damaged cell membranes can restore by themselves. Alkali metals in the electric shock buffer can combine with the cell membranes to promote breakdown of the cell membranes, so that plasmids in the cells are easy to be discharged. An elimination rate of the plasmid reaches 93 %.
Description
Technical field
The invention belongs to the microbial genetics association area.
Technical background
Plasmid be independent of karyomit(e) have outward independently duplicated ability and to the nonessential small-sized virus covalently closed circular double chain DNA molecule of bacteria live, be prevalent in bacterial cell.Owing to being vulnerable to the interference of plasmid DNA when extracting genome, just can obtain the higher genome of purity after the elimination plasmid, this genetics and molecular biology for the research bacterium is significant; Simultaneously, people are in research during with the bacterial strain of plasmid, total hope obtains corresponding plasmid and eliminates bacterium, eliminating the bacterium of plasmid and the original strain of plasmid existence fully compares, genetics function that can the observational study plasmid, the bacterium of eliminating plasmid fully can also as genetic engineering bacterium, be widely used in molecular biology research.Although some plasmid can be lost naturally, frequency is very low, is only 10
-2～10
-8, and most plasmid need to process to improve the plasmid elimination factor by physics, chemical process.
At present, the method for plasmid elimination has method of chemical treatment, Physical and pyroprocessing method and exogenous plasmid to replace null method.Chemical treatment reagent commonly used has sodium lauryl sulphate, acridine orange, ethidium bromide, Cumamycin, Vulkamycin. PA-93, Rifampin etc. to improve the plasmid elimination factor.Hohn etc. utilize acridine orange to eliminate the F plasmid of Bacillus coli cells, and acridine orange reaches plasmid by copying of rapid inhibition plasmid and eliminates purpose, but acridine orange is only effective to intestinal bacteria.The DNA gyrase that Cumamycin and Vulkamycin. PA-93 can suppress in Bacillus coli cells is active, and but DNA gyrase catalysis superhelix forms the DNA of double-stranded closed hoop, and the people such as Novick report utilizes Cumamycin can effectively eliminate the Beta-lactamase From Staphylococcus Aureus plasmid.The people such as Danilevskaya find that Cumamycin all has better effects to the elimination of intestinal bacteria source plasmid such as pBR322, pMB9; In addition temperature is improved 5-7 ℃ and can reach the purpose of eliminating plasmid, Carlton etc. are cultured to the logarithmic phase later stage with cell in hot conditions, be forwarded to after redilution that in fresh substratum, high temperature is cultured to the logarithmic phase later stage again, through repeatedly diluting high-temperature cultivation, the plasmid of part cell disappears.Utilize the uncompatibility principle of plasmid, the Plasmid Transformation that sibship is close is in cell, can not stablize and coexist in same Host Strains due to the close plasmid of relationship, make cell in birth process with one of them plasmid loss, also can reach the purpose of eliminating target plasmid.
Also having a kind of new method is electric shocking method, also can effectively eliminate plasmid.When cell was in electric field, potential difference reached a certain threshold value, and the cytolemma part will be breakdown, forms instantaneous hole plasmid is discharged from cell, and after electric field disappeared, the cytolemma of damage again can self-regeneration.Therefore electric shocking method can be used for eliminating the plasmid in bacterium, simultaneously, the alkalimetal ion concentration of electric shock damping fluid is also an important factor of impact electric shock efficient, thereby alkalimetal ion promotes puncturing of cytolemma by being combined with cytolemma, makes the interior plasmid of born of the same parents more easily leak.Electric shocking method has as more effective in factures such as ethidium bromide, high-temperature cultivation methods than conventional plasmid null method, and also has that the plasmid elimination factor is high, simple operation and other advantages.Although there is report to utilize electric shocking method to eliminate escherichia coli plasmid abroad, this report also reckons without the effect that the electric shock damping fluid is eliminated plasmid, and elimination factor is lower.This patent is by adding the electric shock damping fluid before electric shock, and adopts the electric shock condition of optimizing, and improved the plasmid elimination factor, and accuracy rate is high, and bacterial strain is more stable.Eliminate fast intestinal bacteria antibiotics resistance plasmid with electric shock in conjunction with the method for special electric shock damping fluid, also do not have no relevant report both at home and abroad.The method that this patent provides can fast and effeciently be eliminated intestinal bacteria and see the antibiotics resistance plasmid.
Summary of the invention
Eliminate the plasmid method for present existing intestinal bacteria and need to use a large amount of poisonous chemical reagent such as ethidium bromide, acridine oranges etc., perhaps need can effectively eliminate plasmid through repeatedly going down to posterity, and chemical method and high temperature induction method elimination factor all lower.When utilizing cell to be in electric field, when potential difference reaches a certain threshold value, the cytolemma part is breakdown, forms instantaneous hole, and pore size can make the macromole plasmid discharge from cell, and therefore, electric shocking method can be used for eliminating the plasmid in bacterium.The alkalimetal ion concentration of electric shock damping fluid is also an important factor of impact electric shock efficient, thereby alkalimetal ion promotes puncturing of cytolemma by being combined with cytolemma, makes the interior plasmid of born of the same parents more easily leak.
The object of the present invention is to provide a kind of method of effectively eliminating fast intestinal bacteria antibiotics resistance plasmid.The method economical and effective, fast and easy, and elimination factor is high.Namely
The invention provides and a kind of the colibacillary method of antibiotics resistance plasmid is eliminated in shock by electricity rear acquisition of intestinal bacteria.
The invention provides the elimination that a kind of special electric shock damping fluid promotes the antibiotics resistance plasmid.
A kind of the colibacillary method of antibiotics resistance plasmid is eliminated in the shock by electricity rear acquisition of people enterobacteria, its step is as follows:
A, preparation competent cell:
(1) picking contains antibiotics resistance plasmid pK19mobsacB (Schafer A from the LB flat board, Tauch A, Jager W, et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene, 1994, 145 (1): the single bacterium colony of intestinal bacteria 69-73), intestinal bacteria SCS110 for example, JM109, DH5 α bacterial strain list bacterium colony (Stratagene, Heidelberg, Germany), be inoculated into overnight incubation in the LBG substratum that contains 5ml,
(2) under aseptic condition, bacterium is transferred in the centrifuge tube of precooling, aseptic 1.5ml;
(3) the centrifugal 10min of 4000g reclaims thalline, and this step is carried out under 4 ℃;
(4) 15% glycerine re-suspended cell with the 1ml precooling precipitates;
(5) 4 ℃ to discard liquid after the centrifugal 10min of 4000g, the recovery thalline;
(6) repeating step 4, step 5 are respectively once;
(7) at last thalline is resuspended in 10% glycerine of original volume 1/10, and minute is filled in the 1.5ml centrifuge tube, every pipe 90 μ l, and freezing preservation, standby;
B, electric shock are eliminated:
(1) competent cell is placed on ice melt after, add mixing after the 10 special electric shock damping fluids of μ l, and be placed in and place 10min on ice;
(2) competent cell all is transferred in the electric shock cup of 1mm, 1.8KV/5ms shocks by electricity once;
(3) add immediately the SOC substratum of 1ml, 37 ℃ of incubation 30min after the electric shock;
(4) competent cell is applied to respectively in LBG flat board and kalamycin resistance flat board;
(5) with flat-plate inverted postpone in 37 ℃ of cultivations, after 24h, the dull and stereotyped and kalamycin resistance flat board of LBG all grows single bacterium colony;
C, picking plasmid are eliminated bacterium:
(1) with the toothpick of sterilization, with the bacterium colony that grows on the LBG flat board, be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight;
(2) will grow on the LBG flat board and the bacterium colony of not growing on the kalamycin resistance flat board is chosen, then be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight;
(3) choose single bacterium colony of only growing and not growing on the LBG flat board on the kalamycin resistance flat board;
(4) choose single bacterium colony after, extract plasmid.The plasmid extraction method is referring to alkaline lysis method (Sambrook J in molecular cloning, Fritsch EF, Maniatis T.Molecular Cloning:A laboratory manual, Cold Spring Harbor Laboratory Press.New York, 1989.), whether agarose gel electrophoresis detects plasmid and eliminates;
Described intestinal bacteria comprise intestinal bacteria SCS110, JM109, DH5 α.Eliminable antibiotics resistance plasmid comprises the plasmid pK19mobsacB take intestinal bacteria as the host.
The invention provides a kind of special electric shock damping fluid and promote plasmid to eliminate, this electric shock buffer formulation is:
KCl 0.5M
CaCl
2 0.15M
MgCl
2 0.25M
K
+, Ca
2+, Mg
2+These several positively charged parts all can be combined with electronegative cytolemma, thereby promote puncturing of cytolemma, make the interior plasmid of born of the same parents more easily leak.
The present invention compared with prior art has the following advantages and effect:
(1) the invention provides and a kind of the colibacillary method of antibiotics resistance plasmid is eliminated in shock by electricity rear acquisition of intestinal bacteria, intestinal bacteria are eliminated the plasmid method and need to use a large amount of poisonous chemical reagent such as ethidium bromide, acridine oranges etc. at present, perhaps need through repeatedly going down to posterity, can effectively eliminate plasmid, not only length consuming time, and chemical method and high temperature induction method elimination factor are all lower.Electric shocking method has more effective than conventional plasmid null method, and also has that the plasmid elimination factor is high, simple operation and other advantages.
(2) the present invention improves a kind of special electric shock damping fluid, the alkalimetal ion concentration of electric shock damping fluid is an important factor of impact electric shock efficient, thereby alkalimetal ion promotes puncturing of cytolemma by being combined with cytolemma, makes the interior plasmid of born of the same parents more easily leak.Utilize the electric shock damping fluid to can further improve the plasmid elimination factor.
Description of drawings
Fig. 1 be alkaline lysis respectively extracting intestinal bacteria SCS110, JM109, DH5 α bacterial strain plasmid pK19mobsacB and eliminate plasmid after intestinal bacteria SCS110, JM109,1% agarose gel electrophoresis figure of DH5 α bacterial strain.
M：Lambda DNA/HindIIIMarker；
1: extract pK19mobsacB rear and after the EcoRI enzyme is cut from intestinal bacteria SCS110;
2: extract pK19mobsacB rear and after the EcoRI enzyme is cut from people enterobacteria JM109;
3: extract pK19mobsacB rear and after the EcoRI enzyme is cut from bacillus coli DH 5 alpha;
4: the intestinal bacteria SCS110 after the elimination plasmid;
5: the e. coli jm109 after the elimination plasmid;
6: the bacillus coli DH 5 alpha after the elimination plasmid;
Embodiment
Embodiment 1:
Intestinal bacteria SCS110, JM109, DH5 α contain antibiotics resistance plasmid pK19mobsacB, belong to Gram-negative bacteria, because a kalamycin resistance gene is arranged on the pK19mobsacB plasmid, intestinal bacteria SCS110, JM109, the DH5 α that contains the pK19mobsacB plasmid all can grow containing on the resistant panel of kantlex.
A kind of the colibacillary method of antibiotics resistance plasmid is eliminated in the shock by electricity rear acquisition of people enterobacteria, its step is as follows:
A, preparation competent cell:
(1) picking contains antibiotics resistance plasmid pk19mobsacB (Schafer A from the LB flat board, Tauch A, Jager W, et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene, 1994, 145 (1): the single bacterium colony of intestinal bacteria 69-73), intestinal bacteria SCS110 bacterial strain list bacterium colony (Stratagene for example, Heidelberg, Germany), be inoculated into overnight incubation in the LBG substratum that contains 5ml,
(2) under aseptic condition, bacterium is transferred in the centrifuge tube of precooling, aseptic 1.5ml;
(3) the centrifugal 10min of 4000g reclaims thalline, and this step is carried out under 4 ℃;
(4) 15% glycerine re-suspended cell with the 1ml precooling precipitates;
(5) 4 ℃ to discard liquid after the centrifugal 10min of 4000g, the recovery thalline;
(6) repeating step 4, step 5 are respectively once;
(7) at last thalline is resuspended in 10% glycerine of original volume 1/10, and minute is filled in the 1.5ml centrifuge tube, every pipe 90 μ l, and freezing preservation, standby;
B, electric shock are eliminated:
(1) competent cell is placed on ice melt after, add mixing after the 10 special electric shock damping fluids of μ, and be placed in and place 10min on ice;
(2) competent cell all is transferred in the electric shock cup of 1mm, 1.8KV/5ms shocks by electricity once;
(3) add immediately the SOC substratum of 1ml, 37 ℃ of incubation 30min after the electric shock;
(4) competent cell is applied to respectively in LBG flat board and kalamycin resistance flat board;
(5) with flat-plate inverted postpone in 37 ℃ of cultivations, after 24h, the dull and stereotyped and kalamycin resistance flat board of LBG all grows single bacterium colony;
C, picking plasmid are eliminated bacterium:
(1) with the toothpick of sterilization, with the bacterium colony that grows on the LBG flat board, be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight;
(2) will grow on the LBG flat board and the bacterium colony of not growing on the kalamycin resistance flat board is chosen, then be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight;
(3) choose single bacterium colony of only growing and not growing on the LBG flat board on the kalamycin resistance flat board;
(4) choose single bacterium colony after, extract plasmid.The plasmid extraction method is referring to alkaline lysis method (Sambrook J in molecular cloning, Fritsch EF, Maniatis T.Molecular Cloning:A laboratory manual, Cold Spring Harbor Laboratory Press.New York, 1989.), whether agarose gel electrophoresis detects plasmid and eliminates;
Described intestinal bacteria comprise intestinal bacteria series bacterium: intestinal bacteria SCS110, e. coli jm109, bacillus coli DH 5 alpha.Eliminable plasmid comprises that pK19mobsacB take intestinal bacteria as the host etc. widely contains the plasmid of pBR322 replication origin.
Result is as follows:
Electric shock after mixing with special electric shock damping fluid respectively with competence intestinal bacteria SCS110, e. coli jm109, the bacillus coli DH 5 alpha of exogenous plasmid pK19mobsacB, after swashing through electricity, renewal cultivation is 30 minutes, on that mycin flat board of card-coating and LBG flat board, after 37 ℃ of cultivation 24h, grow 37 single bacterium colonies on intestinal bacteria SCS110 kantlex flat board, grow 454 single bacterium colonies on the LBG flat board, the plasmid elimination factor reaches 91.9%; Grow 54 single bacterium colonies on e. coli jm109 kantlex flat board, grow 597 single bacterium colonies on the LBG flat board, the plasmid elimination factor reaches 91%.Grow 31 single bacterium colonies on bacillus coli DH 5 alpha kantlex flat board, grow 447 single bacterium colonies on the LBG flat board, the plasmid elimination factor reaches 93%.
Choose single bacterium colony of only growing on the LBG flat board and not growing on the kalamycin resistance flat board, whether the alkaline lysis method of extracting plasmid contains plasmid to observe this bacterial strain, and the agarose electrophoresis figure of plasmid such as Fig. 1, swimming lane M are Lambda DNA/HindIII Marker; Swimming lane 1 is for extracting pK19mobsacB rear and after EcoR I enzyme is cut from intestinal bacteria SCS110; Swimming lane 2 is for extracting pK19mobsacB rear and after EcoR I enzyme is cut from e. coli jm109; Swimming lane 3 is for extracting pK19mobsacB rear and after EcoR I enzyme is cut from bacillus coli DH 5 alpha; Swimming lane 4 is the intestinal bacteria SCS110 after the elimination plasmid; Swimming lane 5 is the e. coli jm109 after the elimination plasmid; Swimming lane 6 is the bacillus coli DH 5 alpha after the elimination plasmid; As can be seen from the figure, intestinal bacteria SCS110, JM109, DH5 α plasmid are all eliminated fully.
Claims (3)
1. method of eliminating fast intestinal bacteria antibiotics resistance plasmid, it is characterized in that: with the intestinal bacteria intestinal bacteria that rear acquisition eliminates the antibiotics resistance plasmid of shocking by electricity, and adopt special electric shock damping fluid to promote the elimination of antibiotics resistance plasmid, the basic metal of electric shock damping fluid can promote puncturing of cytolemma, make the interior plasmid of born of the same parents more easily leak, improve the plasmid elimination factor.
2. as claimed in claim 1 with the intestinal bacteria intestinal bacteria that rear acquisition eliminates the antibiotics resistance plasmid of shocking by electricity, its step is as follows:
A, preparation competent cell:
(1) picking contains the single bacterium colony of intestinal bacteria of antibiotics resistance plasmid from the LB flat board, is inoculated into overnight incubation in the LBG substratum that contains 5ml;
(2) under aseptic condition, bacterium is transferred in the centrifuge tube of precooling, aseptic 1.5ml;
(3) the centrifugal 10min of 4000g reclaims thalline, and this step is carried out under 4 ℃;
(4) 15% glycerine re-suspended cell with the 1ml precooling precipitates;
(5) 4 ℃ to discard liquid after the centrifugal 10min of 4000g, the recovery thalline;
(6) repeating step 4, step 5 are respectively once;
(7) at last thalline is resuspended in 10% glycerine of original volume 1/10, and minute is filled in the 1.5ml centrifuge tube, every pipe 90 μ l, and freezing preservation, standby;
B, electric shock are eliminated:
(1) competent cell is placed on ice melt after, add mixing after the 10 special electric shock damping fluids of μ l, and be placed in and place 10min on ice;
(2) competent cell all is transferred in the electric shock cup of 1mm, 1.8KV/5ms shocks by electricity once;
(3) add immediately the SOC substratum (2% (W/V) peptone, 0.5% (W/V) yeast extract, 0.05% (W/V) sodium-chlor, 2.5mM Repone K, 10mM magnesium chloride, 20mM glucose) of 1ml, 37 ℃ of incubation 30min after the electric shock;
(4) competent cell is applied to respectively in LBG flat board and kalamycin resistance flat board;
(5) with flat-plate inverted postpone in 37 ℃ of cultivations, after 24h, the dull and stereotyped and kalamycin resistance flat board of LBG all grows single bacterium colony;
C, picking plasmid are eliminated bacterium:
(1) with the toothpick of sterilization, with the bacterium colony that grows on the LBG flat board, be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight:
(2) will grow on the LBG flat board and the bacterium colony of not growing on the kalamycin resistance flat board is chosen, then be inoculated into respectively the corresponding position in LBG solid plate and kalamycin resistance flat board, incubated overnight;
(3) choose single bacterium colony of only growing and not growing on the LBG flat board on the kalamycin resistance flat board;
(4) choose single bacterium colony after, extract plasmid, whether agarose gel electrophoresis detects plasmid and eliminates;
3. the special electric shock damping fluid of employing as claimed in claim 1, this electric shock buffer formulation is:
KCl 0.5M
CaCl
2 0.15M
MgCl
2 0.25M
K
+, Ca
2+, Mg
2+These several positively charged parts all can be combined with electronegative cytolemma, thereby promote puncturing of cytolemma, make the interior plasmid of born of the same parents more easily leak, and can improve the plasmid elimination factor.
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| CN 201110422779 CN103160493A (en) | 2011-12-16 | 2011-12-16 | Method of rapidly eliminating antibiotic-resistant plasmids in E. coli |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971697A (en) * | 2017-12-27 | 2019-07-05 | 江苏万邦医药科技有限公司 | A method of effectively eliminating Escherichia coli antibiotic resistance plasmids |
| CN112725371A (en) * | 2021-01-19 | 2021-04-30 | 吉林大学 | Method for obtaining ARGs transformants |
-
2011
- 2011-12-16 CN CN 201110422779 patent/CN103160493A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971697A (en) * | 2017-12-27 | 2019-07-05 | 江苏万邦医药科技有限公司 | A method of effectively eliminating Escherichia coli antibiotic resistance plasmids |
| CN109971697B (en) * | 2017-12-27 | 2020-12-08 | 江苏万邦医药科技有限公司 | Method for effectively eliminating escherichia coli antibiotic resistance plasmids |
| CN112725371A (en) * | 2021-01-19 | 2021-04-30 | 吉林大学 | Method for obtaining ARGs transformants |
| CN112725371B (en) * | 2021-01-19 | 2023-03-31 | 吉林大学 | Method for obtaining ARGs transformants |
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Application publication date: 20130619 |