CN103898143A - Simple bacterial gene knockout method - Google Patents
Simple bacterial gene knockout method Download PDFInfo
- Publication number
- CN103898143A CN103898143A CN201210572241.4A CN201210572241A CN103898143A CN 103898143 A CN103898143 A CN 103898143A CN 201210572241 A CN201210572241 A CN 201210572241A CN 103898143 A CN103898143 A CN 103898143A
- Authority
- CN
- China
- Prior art keywords
- gene
- psim19
- colio56
- wfaq
- aseptic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
He invention provides a bacterial gene knockout method. The bacterial gene knockout method comprises following steps: E.coliO56 is conversed with pSIM19 so as to obtain recombinant E.coliO56 containing pSIM19; E.coliO56 electricity competent cells containing pSIM19 are prepared; plasmid pKD4 is taken as a template, kana gene segments containing targeting gene wfaQ homologous arms are amplified via PCR amplification, the kana gene segments containing targeting gene wfaQ homologous arms are inserted into electricity competent cell recombinant bacteria E.coliO56/pSIM19; high-voltage electric shock is carried out, and then SOC fluid medium is added immediately for culturing, and positive recombinants without targeting gene are obtained via selection. The bacterial gene knockout method is very simple, and complicated processes, such as experiments used for determining inductive agent working concentration, are avoided.
Description
Technical field
The invention belongs to biological technical field, concrete relate to a kind of method that easy bacterial gene knocks out.
Background technology
Homologous recombination as the novel method of vivo gene engineering make DNA modification become more easily, more effective, it is the high efficiency method of functional gene group analysis, can in the situation that there is no restriction enzyme and DNA ligase, realize DNA modification.
Tradition homologous recombination method is to utilize RecA recombination system, but take RecA homologous recombination as basic clpp gene is apart from very large deficiency, as target gene homology arm that need to be longer, recombination fraction is very low, is difficult to obtain needed recon etc.1998, Murphy reported first utilize lambda particles phage Red recombination system on escherichia coli chromosome, to carry out the method for Gene Replacement, by the recombination function gene exo of lambda particles phage, bet, gam expresses on multiple copied plasmid, for the Gene Replacement of wild-type E.coli Host Strains.In recent years, Red restructuring is widely used in colibacillary genetic modification with advantages such as its shorter homology arm and higher recombination efficiencies.Red homologous recombination is by the exo of lambda particles phage, bet, 3 genomic constitutions of gam, encode respectively Exo, Beta, 3 kinds of protein of Gam.Exo, as double-strandednucleic acid excision enzyme, can be combined in double-stranded DNA. end, from 5 ' to 3 ' degradation of dna strand, produce 3 ' end strand overhang (3 ' single strand DNA overhangs).Beta is as the strand albumen of annealing, and is combined in to be overhang above by 3 ' end strand of the circumscribed generation of exonuclease (Exo), promotes the annealing of DNA complementary strand.Gam albumen, as the accessory protein of Exo, Beta, can be combined with RecBCD exonuclease, suppresses RecBCD exonuclease activity, suppresses the degraded of the foreign DNA in body.RecBCD is the double-stranded DNA exonuclease that height A TP relies on, the reparation of pitching for homologous recombination and the DNA replication dna of gene in intestinal bacteria.Research shows, Gam albumen is very little for the restraining effect of the ongoing RecBCD double-stranded DNA exonuclease of reaction.In fact, Gam albumen can not suppress an ongoing reaction, but separates the RecBCD that is connected in double-stranded DNA end under the existence of ATP.
Altogether need three kinds of plasmids for the Red recombination system that knocks out bacterial gene: a kind of plasmid is to express Exo, the Beta that recombinase is relevant, the helper plasmid of these 3 kinds of protein of Gam; Another kind of plasmid contains the resistant gene of both sides with Flippases binding site (flipase recognition target, FRT), as pcr template; Last kind of plasmid is that resistant gene is eliminated plasmid, the recombinase energy specific recognition FRT site that it contains, thereby the resistant gene of homologous recombination on elimination karyomit(e).In whole gene knockout process, the most important thing is to control the expression of three required albumen of homology.Express the plasmid of these three albumen as pKD46, pRedET.These two plasmids all contain responsive to temperature type replication origin oriR101, can normal replication in the time cultivating for 37 ℃, and can automatically lose during higher than 37 ℃.In addition, also contain exo, bet, the gam gene of ParaB promoter regulation on pKD46, they pass through L-arabinose abduction delivering, and carry ampicillin resistance gene as selection markers.The promotor of the expression of the upper regulation and control of pRedET exo, bet, gam gene is P
bADpromotor; By L-arabinose abduction delivering equally; But P
bADpromotor is more rigorous than the control methods of ParaB promotor, P
bADpromotor is also subject to just regulation and control and the negative regulation of araC gene product simultaneously, and araC can form mixture with L-arabinose as transcription repressor, transcribes thereby start.
Often select at present Red recombination system for the target gene knocking out in bacterial genomes.The recombination function gene exo of lambda particles phage on different plasmids, bet, the expression regulation mode of gam is different.For the plasmid of selecting by L-arabinose induction recombination function protein expression, knock out the induced concentration that different genes needs to grope L-arabinose, this process takes time and effort.
Summary of the invention
Based on this, the invention provides a kind of method that easy bacterial gene knocks out.
The technical scheme that realizes above-mentioned purpose is as follows.
The method that bacterial gene knocks out, comprises the following steps: (1) pSIM19 Transformed E .coli O56 obtains the restructuring E.coli O56 containing pSIM19;
(3) preparation contains the E.coli O56 electroreception state cell of pSIM19: the positive colony of picking restructuring E.coli O56, cultivate cell concentration OD
600during for 0.4-0.5, put into 42 ℃ of shaking baths, concussion shakes up and is placed on after cooled on ice, centrifugal after, with the aseptic tri-distilled water suspension thalline of precooling, point be filled in the aseptic EP pipe of 2-3 pipe precooling; Then with thalline in the aseptic ultrapure water washing EP pipe of precooling four times, last every pipe adds sterilized water, is prepared into electroreception state cell E.coli O56/pSIM19;
(3), take plasmid pKD4 as masterplate, pcr amplification, with the kana gene fragment of goal gene wfaQ homology arm, adds the described kana gene fragment with goal gene wfaQ homology arm in electroreception state cell recombinant bacterium E.coliO56/pSIM19; After completing, high-voltage electric shock adds immediately SOC liquid nutrient medium, 37 ℃, cultivate after 1.5 ± 0.2h, after centrifugal, outwell part supernatant, get the kalamycin resistance flat board of appropriate thallus suspension liquid coating containing 50 ± 2g/mL, cultivate, grow single bacterium colony, single bacterium colony is lined containing in the kalamycin resistance flat board of 50 ± 2g/mL, grow denseer lawn, screening obtains the positive recombinant that goal gene has been eliminated.
In an embodiment, the step of described pSIM19 Transformed E .coli O56 is: plasmid is joined in E.coli O56 competent cell, mix gently rear ice bath therein; 42 ℃ of water-bath heat shock 90s, are transferred to rapidly on ice, continue ice bath 2 ± 1min; Under aseptic condition, add LB substratum, 37 ℃, 100 ± 10rpm shakes slowly, renewal cultivation 1 ± 0.2h; After centrifugal culture, get appropriate culture coating containing on the solid LB agar plate of 100 ± 2g/mL Amp, after liquid is absorbed by solid medium, flat board is inverted, 37 ± 2 ℃ of constant temperature culture, 12-16h, obtains the restructuring E.coli O56 containing pSIM19.
Therein in an embodiment, in step (2), with in the aseptic ultrapure water washing EP pipe of precooling when thalline, at every turn all at 12000 ± 20rpm, under the condition of 4 ± 1 ℃, centrifugal 1 ± 0.1min is to remove ultrapure water.
In an embodiment, in step (3), pcr amplification is SEQ ID NO.1 and SEQ ID NO.2 with the amplimer of the kana gene fragment of goal gene wfaQ homology arm therein.
In an embodiment, described aseptic ultrapure water is MiliQ ultrapure water therein.
Often select at present Red recombination system for the target gene knocking out in bacterial genomes.The recombination function gene exo of lambda particles phage on different plasmids, bet, the expression regulation mode of gam is different.For the plasmid of selecting by L-arabinose induction recombination function protein expression, knock out the induced concentration that different genes needs to grope L-arabinose, this process takes time and effort.The present invention selects the responsive to temperature type plasmid pSIM19 by temperature-induced recombination function protein expression, gropes this loaded down with trivial details link thereby saved inductor working concentration.Meanwhile, the present invention selects MiliQ ultrapure water to turn damping fluid as linear DNA electricity, and washing is the centrifugal 1min of 12000rpm when thalline, washs 4-5 time, compares to turn damping fluid with 10% glycerine as electricity and saved a lot of time.
Accompanying drawing explanation
Fig. 1 is pSIM19 plasmid map;
Fig. 2 is Fig. 1 bacterium colony PCR checking positive recombinant O56/ Δ wfaQ/kana; Wherein, 1: with wild-type E.coliO56 template; 3-11 and 13: take positive recombinant O56/ Δ wfaQ/kana as template; 2 and 12: take false positive recon as template.
Embodiment
In order more clearly to understand technology contents of the present invention, be described with reference to the accompanying drawings especially exemplified by following examples.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.In embodiment, various conventional chemical reagent used, are commercially available prod.
The plasmid pSIM19 that the present invention selects is presented by Donald L Court laboratory, and this plasmid can gratuitously obtain from many laboratories.Referring to document: Sharan K S, et al.Recombineering:a homologousrecombination-based method of genetic engineering.Nat Pro.2009 (4)., as Donald LCourt (
ncifcrf.gov).Those skilled in the art also can, according to prior art, build and obtain according to collection of illustrative plates voluntarily.Its collection of illustrative plates is asked for an interview Fig. 1.
Embodiment mono-: knock out the gene wfaQ in E.coli O56 with RED recombination system.
One, pSIM19 Transformed E .coli O56
1. 2L plasmid is joined in 100L E.coli O56 competent cell, mix gently rear ice bath 30min;
2. 42 ℃ of water-bath heat shock 90s, are transferred to rapidly on ice, continue ice bath 2min;
3. under aseptic condition, add 900L LB substratum, 37 ℃, 100rpm shakes slowly, renewal cultivation 1h;
4. the centrifugal culture of 5000rpm, gets appropriate culture coating containing on the solid LB agar plate of 100g/mL Amp after 5min, after liquid is absorbed by solid medium, flat board is inverted, and 37 ℃ of constant temperature culture 12-16h, obtain the restructuring E.coli O56 containing pSIM19.
Two, preparation is containing the E.coli O56 electroreception state cell of pSIM19
The positive colony of picking restructuring E.coli O56 (containing temperature sensitivity plasmid pSIM19), 30 ℃, 200rpm incubated overnight.Next day, get 1% overnight culture switching 50mL SOB liquid nutrient medium (formula of this substratum is: by 2g Tryptones, 0.5g yeast powder, 0.0585g sodium-chlor, 0.0186g Repone K is dissolved in pure water, after mixing, is settled to 100mL, and 121 ℃ of moist heat sterilization 20min are for subsequent use.), add 500L concentration is the aseptic MgCl of 1M simultaneously
2, treat cell concentration OD
600for putting it in 42 ℃ of shaking baths after 0.4-0.5 (2h approximately grows), 200rpm shakes 15min, then at once shaking flask is placed on ice, starts to make electroreception state cell after 5-10min.4 ℃, after the centrifugal 7min of 12000rpm, with the aseptic tri-distilled water suspension thalline of 2mL precooling, divide and be filled in the aseptic 1.5mLEP pipe of 2-3 pipe precooling.Then use in aseptic ultrapure water (MiliQ ultrapure water) the washing pipe of precooling thalline four times (at every turn all at 12000rpm, under the condition of 4 ℃, centrifugal 1min is to remove ultrapure water), it is 50-60L that last every pipe adds sterilized water to final volume, and the electroreception state cell (called after E.coliO56/pSIM19) of preparation can be deposited one week in-80 ℃ thus.
Three, turn in above-mentioned electroreception state cell E.coliO56/pSIM19 with the resistant gene electricity of wfaQ homology arm
First take plasmid pKD4 (commercialization plasmid) as masterplate, take primer O56-wfaQ-k-F/wfaQ-k-R, in order to knock out the method for primer (sequence is in table 1) by polymerase chain reaction PCR, (PCR reaction system please refer to the reaction system that fermentas article No. provides as #EP0502 (Pfu DNA polymerase); This experiment amplification condition is 98 ℃ of denaturation 2min, 98 ℃ of sex change 30s, and 56 ℃ of annealing 30s, 72 ℃ are extended 1.5min, totally 30 circulations; Last 72 ℃ are extended 5min.) increase with the kana gene fragment of goal gene wfaQ homology arm.Be that the gene fragment of 200-300ng/L (is above-mentionedly (to be for No. GeneBank: DQ220293.1) the kana gene fragment of homology arm (kantlex nucleotidyl transferase gene with goal gene wfaQ to adding 40L concentration in electroreception state cell recombinant bacterium E.coli O56/pSIM19, it is for No. GeneBank: NC_002952)), in the electric revolving cup that the aseptic thickness that is transferred to precooling after mixing is 2mm, after completing, 2.5kV high-voltage electric shock adds immediately 1mL SOC liquid nutrient medium (having added final concentration is the SOB liquid nutrient medium of 20mM glucose), 37 ℃, after 100rpm renewal cultivation 1.5h, after the centrifugal 5min of 6000rpm, outwell part supernatant, get the kalamycin resistance flat board (containing final concentration be the agarose solid medium of 50g/mL kantlex) of appropriate thallus suspension liquid coating containing 50g/mL.Flat board is put in 37 ℃ of incubators, and about 16-20h grows single bacterium colony, and single bacterium colony is lined in the kalamycin resistance flat board containing 50g/mL, grows denseer lawn after 12h.By bacterium colony PCR, (it is the reaction system that AP111 (EasyTaq DNA polymerase) provides that PCR reaction system please refer to Transgene article No.; This experiment amplification condition is 94 ℃ of denaturation 4min, 94 ℃ of sex change 30s, and 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 5min.) method identified judges the situation that knocks out of goal gene, agarose gel electrophoresis figure asks for an interview Fig. 2.
In E.coli O56 genome, knocking out between primer O56-wfaQ-k-F/wfaQ-k-R at a distance of 1149bp shown in table 1, detect between primer wfaQ-t-F/wfaQ-t-R (sequence is in table 1) at a distance of 1250bp, therefore, while carrying out bacterium colony PCR checking O 56/ Δ wfaQ/kana positive recombinant with detection primer, should be 1250bp take wild-type O56 (negative control) bacterium liquid as the PCR product size of template; And the kana resistant gene that to knock out between primer due to the size of having recombinated be 1496bp of positive recombinant O56/ Δ wfaQ/kana, therefore should be at a distance of 1760bp between the detection primer of positive recombinant.So, to detect primer wfaQ-t-F/wfaQ-t-R as primer, with the negative contrast of E.coli O56, by bacterium colony PCR checking positive recombinant O56/ Δ wfaO/kana, electrophoresis result as shown in Figure 2, be numbered 3-11 and 13 positive recons, the goal gene wfaQ in E.coli O56 is eliminated.
Table 1: amplimer
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. the method that bacterial gene knocks out, is characterized in that, comprises the following steps:
(1) pSIM19 Transformed E .coliO56, obtains the restructuring E.coliO56 containing pSIM19;
(2) preparation contains the E.coliO56 electroreception state cell of pSIM19: the positive colony of picking restructuring E.coli O56, cultivate cell concentration OD
600during for 0.4-0.5, put into 42 ℃ of shaking baths, concussion shakes up and is placed on after cooled on ice, centrifugal after, with the aseptic tri-distilled water suspension thalline of precooling, point be filled in the aseptic EP pipe of 2-3 pipe precooling; Then with thalline in the aseptic ultrapure water washing EP pipe of precooling three to five times, last every pipe adds sterilized water, is prepared into electroreception state cell E.coliO56/pSIM19;
(3), take plasmid pKD4 as masterplate, pcr amplification, with the kana gene fragment of goal gene wfaQ homology arm, adds the described kana gene fragment with goal gene wfaQ homology arm in electroreception state cell recombinant bacterium E.coli O56/pSIM19; After completing, high-voltage electric shock adds immediately SOC liquid nutrient medium, 37 ± 0.5 ℃, cultivate after 1.5 ± 0.2h, after centrifugal, outwell part supernatant, get the resistant panel of appropriate thallus suspension liquid coating containing the kantlex of 50 ± 2g/mL, cultivate, grow single bacterium colony, single bacterium colony is lined in the resistant panel of the kantlex that contains 50 ± 2g/mL, grow denseer lawn, screening obtains the positive recombinant that goal gene has been eliminated.
2. the method that bacterial gene according to claim 1 knocks out, is characterized in that, the step of described pSIM19 Transformed E .coli O56 is: plasmid is joined in E.coliO56 competent cell, mix gently rear ice bath; 42 ℃ of water-bath heat shock 90 ± 2s, are transferred to rapidly on ice, continue ice bath 2 ± 1min; Under aseptic condition, add LB substratum, 37 ± 0.5 ℃, 100 ± 10rpm shakes slowly, renewal cultivation 1 ± 0.2h; After centrifugal culture, get appropriate culture coating containing on the solid LB agar plate of 100 ± 2g/mL Amp, after liquid is absorbed by solid medium, flat board is inverted, 37 ± 0.5 ℃ of constant temperature culture, 12-16h, obtains the restructuring E.coliO 56 containing pSIM19.
3. the method that bacterial gene according to claim 1 knocks out, it is characterized in that, with in the aseptic ultrapure water washing EP pipe of precooling when thalline, at every turn at 12000 ± 20rpm, under the condition of 4 ± 1 ℃, centrifugal 1 ± 0.1min is to remove ultrapure water in step (2).
4. the method knocking out according to the bacterial gene described in claim 1-3 any one, is characterized in that, described aseptic ultrapure water is MiliQ ultrapure water.
5. the method knocking out according to the bacterial gene described in claim 1-3 any one, is characterized in that, in step (3), pcr amplification is SEQ IDNO.1 and SEQ ID NO.2 with the amplimer of the kana gene fragment of goal gene wfaQ homology arm.
6. the positive recombinant E.coliO56 that the preparation-obtained goal gene wfaQ of method knocking out according to the bacterial gene described in claim 1-5 any one is knocked.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210572241.4A CN103898143A (en) | 2012-12-25 | 2012-12-25 | Simple bacterial gene knockout method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210572241.4A CN103898143A (en) | 2012-12-25 | 2012-12-25 | Simple bacterial gene knockout method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103898143A true CN103898143A (en) | 2014-07-02 |
Family
ID=50989711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210572241.4A Pending CN103898143A (en) | 2012-12-25 | 2012-12-25 | Simple bacterial gene knockout method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103898143A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107641631A (en) * | 2017-09-07 | 2018-01-30 | 浙江工业大学 | A kind of method that bacillus coli gene is knocked out based on CRISPR/Cas9 systems by chemical conversion mediation |
| CN113943748A (en) * | 2021-11-05 | 2022-01-18 | 华南农业大学 | Recombination system in pseudomonas syringae and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094036A (en) * | 2010-12-13 | 2011-06-15 | 西北农林科技大学 | Allele double knockout targeting vector system and construction method thereof |
| CN102154188A (en) * | 2010-12-22 | 2011-08-17 | 中国人民解放军第三军医大学 | nfi-gene-knocked-out mutant strain of escherichia coli DH5 alpha as well as preparation method and application thereof |
-
2012
- 2012-12-25 CN CN201210572241.4A patent/CN103898143A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094036A (en) * | 2010-12-13 | 2011-06-15 | 西北农林科技大学 | Allele double knockout targeting vector system and construction method thereof |
| CN102154188A (en) * | 2010-12-22 | 2011-08-17 | 中国人民解放军第三军医大学 | nfi-gene-knocked-out mutant strain of escherichia coli DH5 alpha as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 党利君: "酶法合成唾液酸化寡糖及细菌唾液酸转移酶WfaN的功能鉴定", 《万方数据》, 30 November 2012 (2012-11-30) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107641631A (en) * | 2017-09-07 | 2018-01-30 | 浙江工业大学 | A kind of method that bacillus coli gene is knocked out based on CRISPR/Cas9 systems by chemical conversion mediation |
| CN113943748A (en) * | 2021-11-05 | 2022-01-18 | 华南农业大学 | Recombination system in pseudomonas syringae and application |
| CN113943748B (en) * | 2021-11-05 | 2023-06-16 | 华南农业大学 | Recombination system in pseudomonas syringae and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104404036B (en) | Conditional gene knockout method based on CRISPR/Cas9 technologies | |
| CN110527697B (en) | RNA fixed-point editing technology based on CRISPR-Cas13a | |
| CN105671080B (en) | Method for sheep MSTN gene knockout and site-specific integration exogenous gene mediated by CRISPR-Cas9 system | |
| RU2020102451A (en) | NUCLEIC ACID DIRECTED NUCLEASE | |
| CN104651401A (en) | Method for knocking out two mir-505 alleles | |
| CN102703424B (en) | A kind of method of genome of E.coli point mutation of recombined engineering mediation | |
| CN107151677B (en) | Method for knocking out low transfection efficiency cell line based on CRISPR/Cas9 multiple genes | |
| AU2016260738B2 (en) | IrrE protein functional domain for improving anti-oxidation capability of cell and application thereof | |
| CN101948871B (en) | Marine microalgae chloroplast expression vector and application thereof | |
| CN111849979A (en) | sgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line | |
| CN104371993A (en) | Asparaginase mutant with enhanced enzyme activity | |
| CN103898143A (en) | Simple bacterial gene knockout method | |
| WO2013144663A2 (en) | Method of determination of neutral dna sequences in the genome, system for targeting sequences obtained thereby and methods for use thereof | |
| CN103849639B (en) | A kind of method improving halfcystine utilization ratio biosynthesis of glutathione | |
| CN113025613A (en) | ADORA2A gene knockout cell and construction method and application thereof | |
| CN110129228B (en) | Preparation method of nocardia competent cells and nocardia gene editing method | |
| CN103352050B (en) | Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination | |
| CN113881678B (en) | C/EBPZ gene promoter and application thereof | |
| CN116376945A (en) | I-type caspase gene insertion tool and application | |
| CN103232994B (en) | Method for screening unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans | |
| CN111321154A (en) | Method for regulating ganoderic acid yield based on higher fungus ganoderma function gene editing | |
| AU2020204574A1 (en) | B.licheniformis engineering bacteria for inhibiting bacterial autolysis, construction method and application thereof | |
| Chen et al. | Multiple-copy-gene integration on chromosome of Escherichia coli for beta-galactosidase production | |
| CN104630272A (en) | Demethylation-based vector for promoting self-renewal and proliferation of germline stem cells and application thereof | |
| EP3847252A1 (en) | The gene therapy dna vector gdtt1.8nas12 and the method for obtaining thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140702 |
|
| RJ01 | Rejection of invention patent application after publication |