CN104450765A - Integron In1069 - Google Patents
Integron In1069 Download PDFInfo
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- CN104450765A CN104450765A CN201410640497.3A CN201410640497A CN104450765A CN 104450765 A CN104450765 A CN 104450765A CN 201410640497 A CN201410640497 A CN 201410640497A CN 104450765 A CN104450765 A CN 104450765A
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Abstract
The invention discloses an integron In1069 with a sequence as shown in SEQ ID NO.1. The integron is found in a genome of drug-resistant Escherichia coli EC6335. The integron contains a plurality of drug-resistant gene cassettes. Therefore, due to the discovery of the integron disclosed by the invention, a certain guiding effect is provided for clinical medication, some clinically-used antibacterial agents can be favorably reduced, the selective pressure for horizontal transfer of the drug-resistant gene cassettes is reduced, and the outbreak of drug-resistant bacteria is avoided.
Description
Technical field
The invention belongs to Protocols in Molecular Biology and drug-resistant bacteria monitoring field, relate to a kind of newfound integron In1069 closely-related with Drug Resistance of E. coli.
Background technology
Intestinal bacteria are typical gram negative bacilluses, and its colibacillosis caused is a kind of common disease.The main prophylactico-therapeutic measures of colibacillosis is application vaccine and microbiotic.But along with antibiotic extensive, lasting and improper application, intestinal bacteria Antibiotic Resistance constantly expands, resistance levels improves constantly, and intestinal bacteria resistance and multidrug resistant phenomenon are very serious, bring potential harm to the development of livestock industry and human health.
Integron (integron) is the intergrase identification by self coding, catches, integrates or shear extracellular episome or gene fragment, and makes it the recombinant expression system being converted into functioning gene.Integron can be divided into multiple different type according to the difference of intergrase sequence.I class integron to find in gram-negative bacteria extensively to distribute so far, recall rate is maximum.The basic structure of I class integron is made up of 3 parts, two ends are highly conserved sequences, be called 5 ' conservative end (5 '-CS) and 3 ' conservative end (3 '-CS), 5 '-CS is about 1.4kb, 3 '-CS is about 2kb, region between 5 '-CS and 3 '-CS is called variable region, and variable region is made up of one or more box gene, and box gene is generally containing Drug-resistant genes.Integron is a kind of moveable Genetic elements; on the plasmid being positioned at bacterium or karyomit(e), multiple Drug-resistant genes can be carried, and efficient, the fast transfer of drug resistant gene can be caused; cause the diffusion between of the same race or different strain, thus express the antibiotic multi-drug resistant of difference.
It is integrons of bacteria molecular evolution and the major reason of catching more Multidrug resistance that clinical antibacterials are widely applied, therefore the reasonable application of antibacterials should be strengthened, to reduce, integron occurs, the outside induced environment of molecular evolution, delays the generation of multidrug resistance bacterial strain.
Summary of the invention
The invention provides a kind of integron containing drug resistant gene box, by its called after In1069, its sequence is as shown in SEQ ID NO.1.This integron contains aacA4 '-3 box gene, bla
iMP-1box gene.Above-mentioned integron causes the bacterial strain EC6335 containing this integron to have resistance to Multiple Classes of Antibiotics medicine.These microbiotic comprise: aminoglycoside antibiotics, β-lactam antibitics.
Common aminoglycoside antibiotics comprises: Streptomycin sulphate, gentamicin, kantlex, tobramycin, amikacin.
Common beta-lactam class microbiotic comprises: penicillin, cynnematin, carbapenems, and other atypia β-lactam antibiticss such as the cephamycin-type of new development, sulfomycin class, monobactams.
Integron of the present invention obtains by the following method:
(1) cultivate at municipal hospital intensive care room, Taizhou patient's fester in the sample of the positive and isolate the bacterium of 1 strain to cephalo pyridine, imipenem-resistant;
(2) VITEK 2 and the dull and stereotyped agar diffusion method of susceptibility (supplementing drug sensitive test paper) is then used by the mono-clonal bacterial strain of separation to carry out Bacteria Identification and drug sensitive test, through identifying that this bacterial strain is intestinal bacteria, by its called after EC6335 in operation sequence; Meanwhile, test confirms that this bacterial strain has remarkable resistance to Multiple Classes of Antibiotics; Described microbiotic is aztreonam, amikacin, tobramycin, butylamine OK a karaoke club, gentamicin, cefotetan, Cephazolin, cefepime, ceftazime, ceftriaxone, imipenum, piperacillin/Tazobactam Sodium, Sulbactam/Cefoperazone (sulperazone);
(3) EC6335 strain culturing is increased, extract the plasmid of described Resistant strain;
(4) DNA fragmentation on the plasmid that obtains through step (3) of amplification, order-checking;
(5) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify the integron of sequence as shown in SEQ ID NO.1.
Further, the concrete operation step of step (4) is as follows: the plasmid obtained with step (3), for template, utilizes the primer in table 1 to carry out DNA fragmentation amplification:
Table 1 primer sequence
Note: * RP represents replication protein gene; * HP represents putative protein gene; * RIP represents and copies startup protein gene
Above-mentioned PCR primer is carried out agarose gel electrophoresis, reclaim all fragments amplified, be connected respectively by amplified fragments in conventional manner with pMD19-T carrier, then heat-shock transformed method proceeds in competent cell E.coliTOP10, positive transformant is selected, order-checking with blue hickie experiment.
Present invention also offers a kind of recombinant plasmid containing integron In1069 sequence.Described plasmid comprises integron In1069 sequence.In specific embodiment of the present invention, described recombinant plasmid is recombinated by integron In1069 sequence and pET32a carrier and is formed, and integron insertion point is BamHI restriction enzyme site place on pET32a carrier.The empty carrier that described recombinant plasmid uses can use any common plasmid vector to replace.The method of described recombinant plasmid is also ordinary method well known to those skilled in the art.
Present invention also offers a kind of joint bacterial strain containing integron In1069.In order to study the resistance of integron, the present invention utilizes bacterial plasmid transduction experiment to carry out the function of Testing and appraisal integron In1069, by EC6335 bacterial strain and E.coli J53 AzR (to sodiumazide resistance) bacterial strain Dual culture, by containing sodiumazide (300mg/L) and imipenum (2mg/L) plate screening zygote, zygote is done drug sensitive test, and recipient bacterium has the resistance of donor bacterium.Wherein, imipenum used can replace with agents: amikacin, Ampicillin Trihydrate, ampicillin/sulbactam, aztreonam, Kefzol, cefepime, cefotetan, ceftazime, ceftriaxone, ertapenem, gentamicin, piperacillin/Tazobactam Sodium, tobramycin, Sulbactam/Cefoperazone.
Present invention also offers a kind of recombinant bacterial strain containing integron In1069.For confirming that the resistance of above-mentioned joint bacterial strain derives from the integron in EC6335 bacterial strain further, this integron In1069 is cloned in pET32a carrier by the present invention, and integron insertion point is BamHI restriction enzyme site place on pET32a carrier.And be transformed in wild-type E.coli JM109, select positive colony (bacterium containing pET32a-integron) by the flat board containing Amp.Positive colony is done drug sensitive test, and the integron In1069 in result display EC6335 bacterial strain can make restructuring E.coli JM109 bacterial strain to following Drug-resistant: amikacin, Ampicillin Trihydrate, ampicillin/sulbactam, aztreonam, Kefzol, cefepime, cefotetan, ceftazime, ceftriaxone, ertapenem, gentamicin, imipenum, piperacillin/Tazobactam Sodium, tobramycin, Sulbactam/Cefoperazone.
Integron sequence of the present invention can business be synthesized, and preferably, integron In1069 sequence is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
Advantage of the present invention and beneficial effect:
(1) Late Cambrian of the present invention sequence is the integron In1069 that comprises multidrug resistant gene box of SEQ ID NO.1, and the nucleotide sequence of this integron has no report in the prior art.
(2) discovery of integron sequence of the present invention, for clinical application provides certain directive function.Be conducive to the use reducing some antibacterials clinically, reduce the selective pressure of such drug resistant gene box horizontal transfer, prevent the Outbreak of drug-resistant bacteria.
Accompanying drawing explanation
Fig. 1 is the structural representation of integron In1069 of the present invention.
Concrete embodiment
Further illustrate the present invention below in conjunction with specific embodiment, embodiments of the invention only for explaining the present invention, and do not mean that and limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The qualification of embodiment 1 integron In1069
1, the separation andpreconcentration of EC6335 bacterial strain
1.1 material
Bacterial susceptibility card: the AST-GN13 of French bio Merieux.AST-GN13 susceptibility kind has: amikacin, Ampicillin Trihydrate, ampicillin/sulbactam, aztreonam, Kefzol, cefepime, cefotetan, ceftazidime, ceftriaxone, Ciprofloxacin, ertapenem, gentamicin, imipenum, levofloxacin, furadantin, piperacillin/Tazobactam Sodium, tobramycin, SMZ.
Supplement drug sensitive test paper (the dull and stereotyped agar diffusion experiment of susceptibility): the scraps of paper derive from Oxoid company of Britain, have Sulbactam/Cefoperazone (75 μ g/30 μ g).
1.2 method
Instrumental: municipal for Taizhou hospital intensive care room patient blood is cultivated positive bacterial strain transferred species to separation and Culture on blood agar (at 35 DEG C containing 5%CO
2cultivate 16-18h in incubator), then by be separated pure bacterium in operation sequence on VITEK 2 machine do Bacteria Identification and drug sensitive test.
Supplement drug sensitive test: spread upon on MH flat board by the bacterium liquid of 0.5 Maxwell unit, stick the Sulbactam/Cefoperazone scraps of paper in operation sequence, at 35 DEG C containing 5%CO
2after cultivating 16-18h in incubator, by CLSI 2012 standard identification and susceptibility result.
1.3 result
1.3.1 instrumental result
Be accredited as intestinal bacteria through VITEK 2 microbiological analysis instrument, identify that probability is 99%, by its called after EC6335.
1.3.2 drug sensitive experiment result
Drug sensitive experiment result shows, EC6335 bacterial strain produces resistance for following medicine: aztreonam, amikacin, tobramycin, butylamine OK a karaoke club, gentamicin, cefotetan, Cephazolin, cefepime, ceftazime, ceftriaxone, imipenum, piperacillin/Tazobactam Sodium, Sulbactam/Cefoperazone (sulperazone).
2, the acquisition of integron In1069 and qualification
2.1 method
2.1.1EC6335 the plasmid extraction of bacterial strain
Adopt the plasmid of TaKaRa to extract test kit on a small quantity, operation steps extracts bacteria plasmid DNA to specifications, and as pcr template ,-20 DEG C of Refrigerator stores are for subsequent use.
2.1.2 integron sequence amplification
With the plasmid of said extracted for template, the primer in table 1 is utilized to carry out DNA fragmentation amplification.
Above-mentioned PCR primer is carried out agarose gel electrophoresis, reclaim all fragments amplified, be connected respectively by amplified fragments in conventional manner with pMD19-T carrier, then heat-shock transformed method proceeds in competent cell E.coliTOP10, positive transformant is selected, order-checking with blue hickie experiment.
2.1.3 sequence assembly
Utilize ContigExpress Program software to carry out sequence assembly, analytical structure the above-mentioned DNA fragmentation amplified, identify the integron of sequence as shown in SEQ ID NO.1, its structural representation as shown in Figure 1.
The function of embodiment 2 plasmid transduction experimental study integron In1069
1, method
(1) donor bacterium is EC6335 bacterial strain, and recipient bacterium is E.coli J53AzR (to sodiumazide resistance).Donor bacterium, recipient bacterium are inoculated in respectively on LB flat board, 35 DEG C of incubated overnight.The single bacterium colony of picking is inoculated in the LB meat soup of 4mL respectively, and 37 DEG C of 220r/min shake bacterium and are cultured to logarithmic phase.Respectively get 0.5ml confession, recipient bacterium in the LB meat soup of 4ml, 37 DEG C of left undisturbed overnight are cultivated.Zygote, with the screening of pancreas soy agar (TSA) plate, contains sodiumazide (300mg/L) and imipenum (2mg/L) in flat board.Put 35 DEG C and hatch 18-24h.The plasmid (step is with embodiment 1) extracting Resistant strain is pcr amplification template, and the primer in use table 1 detects intI1, tnpA, aacA4 '-3, whether * RIP, Orf44, IMP-1, TniC gene fragment exists.
(2) choose the positive strain comprising said gene fragment and carry out drug sensitive experiment, step is with embodiment 1.
2, result
Drug sensitive experiment result shows, the drug sensitive reaction that integron In1069 sequence expresses positive strain is identical with EC6335 bacterial strain, represent integron In1069 sequence conjugal transfer success, and this integron result in conjugative bacteria creates resistance to agents: amikacin, Ampicillin Trihydrate, ampicillin/sulbactam, aztreonam, Kefzol, cefepime, cefotetan, ceftazime, ceftriaxone, ertapenem, gentamicin, imipenum, piperacillin/Tazobactam Sodium, tobramycin, Sulbactam/Cefoperazone.
The function of embodiment 3 gene recombination experimental study integron In1069
1, method
(1) integron In1069 sequence is synthesized: integron In1069 sequence is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(2) the integron In1069 of synthesis is connected with pMD18T carrier, connect product to add in the competence of 100 μ lE.coli JM109 and transform, cultivating containing on the flat board of IPTG, x-gal, Amp, IPTG, x-gal, Amp resistant strain is extracted plasmid, and PCR detects integron In1069 and whether inserts (step is with embodiment 2) in genome.Then by integron In1069 sequence clone in pET32a carrier, insertion point BamHI restriction enzyme site place on pET32a carrier of integron, conventionally prepares, and obtains the pET32a recombinant plasmid containing integron In1069 sequence.By recombinant plasmid transformed in competence E.coli JM109 cell.
(2) Resistance detection is carried out to the clone bacterium successfully constructed
The dull and stereotyped agar diffusion method of VITEK 2 Bacteria Identification of French bioMerieux and Analysis of Drug Susceptibility system and susceptibility (supplementing drug sensitive test paper) is adopted to carry out Resistance detection to recombinant bacterium E.coli JM109 (pET32a-integron In1069).
2, result
Drug sensitive experiment result shows, the drug sensitive reaction of recombinant bacterium E.coli JM109 (pET32a-integron In1069) is identical with EC6335 bacterial strain, represent integron In1069 sequence Successful integration in the genome of E.coli JM109, and this integron result in recombinant bacterium E.coli JM109 creates resistance to agents: amikacin, Ampicillin Trihydrate, ampicillin/sulbactam, aztreonam, Kefzol, cefepime, cefotetan, ceftazime, ceftriaxone, ertapenem, gentamicin, imipenum, piperacillin/Tazobactam Sodium, tobramycin, Sulbactam/Cefoperazone.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (9)
1. an integron, is characterized in that, described integron sequence as shown in SEQ ID NO.1, described integron called after In1069.
2. a recombinant plasmid, is characterized in that, comprises integron according to claim 1 in described recombinant plasmid.
3. recombinant plasmid according to claim 2, is characterized in that, the vector plasmid used in the preparation process of described recombinant plasmid is pET32a carrier.
4. a conjugative bacteria, is characterized in that, containing integron according to claim 1 in described conjugative bacteria; Described conjugative bacteria is prepared by EC6335 bacterial strain and E.coli J53 AzR bacterial strain.
5. a recombinant bacteria, is characterized in that, containing integron according to claim 1 in described recombinant bacteria; Described recombinant bacteria is proceeded in E.coli JM109 bacterial strain by the recombinant plasmid described in Claims 2 or 3 to be prepared from.
6. the acquisition methods of integron according to claim 1, is characterized in that, said method comprising the steps of:
(1) cultivate at municipal hospital intensive care room, Taizhou patient's fester in the sample of the positive and isolate the bacterium of 1 strain to cephalo pyridine, imipenum;
(2) the mono-clonal bacterial strain that step (1) is separated is used in operation sequence VITEK 2 and the dull and stereotyped agar diffusion method of susceptibility, supplement drug sensitive test paper simultaneously and carry out Bacteria Identification and drug sensitive test, through identifying that this bacterial strain is Klebsiella pneumonia, called after EC6335;
(3) EC6335 strain culturing is increased, extract the plasmid of described EC6335 bacterial strain;
(4) DNA fragmentation on the plasmid that obtains through step (3) of amplification, order-checking;
(5) utilize ContigExpress Program software to carry out sequence assembly, obtain the structure of integron according to claim 1.
7. the preparation method of a conjugative bacteria according to claim 4, it is characterized in that, the concrete operation step of described preparation method is as follows: by EC6335 bacterial strain and E.coli J53 AzR bacterial strain Dual culture, by the plate screening zygote containing sodiumazide and EC6335 bacterial strain tolerance medicine.
8. preparation method according to claim 7, is characterized in that, described EC6335 bacterial strain tolerance medicine is imipenum.
9. the preparation method of a recombinant bacteria according to claim 5, it is characterized in that, the concrete operation step of described preparation method is as follows: be cloned into by integron In1069 in pET32a carrier, then recombinant plasmid transformed is entered in wild-type E.coli JM109, select positive colony by the flat board containing Amp.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862327A (en) * | 2015-05-18 | 2015-08-26 | 王冬国 | Novel integron containing multiple drug-resistant gene cassettes |
| CN104878037A (en) * | 2015-05-18 | 2015-09-02 | 王冬国 | Integron In1085 |
| CN105039357A (en) * | 2015-07-09 | 2015-11-11 | 王冬国 | Plasmid fragment carrying novel gene qepA3 |
| CN106047898A (en) * | 2016-05-10 | 2016-10-26 | 王冬国 | Novel integron In1289 containing multiple drug-resistant gene cassettes |
| CN106047900A (en) * | 2016-05-10 | 2016-10-26 | 王冬国 | Novel integron In1287 |
| CN106167802A (en) * | 2016-05-10 | 2016-11-30 | 王冬国 | A kind of novel integron In1290 |
| CN109913399A (en) * | 2019-04-12 | 2019-06-21 | 贵州省水产研究所 | A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application |
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| CN101503670A (en) * | 2008-02-04 | 2009-08-12 | 复旦大学附属华山医院 | Engineering bacterial strain containing integron |
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- 2014-11-13 CN CN201410640497.3A patent/CN104450765A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101503670A (en) * | 2008-02-04 | 2009-08-12 | 复旦大学附属华山医院 | Engineering bacterial strain containing integron |
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| NCBI: "GenBank: HQ651093.1", 《NCBI GENBANK》 * |
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| 杜艳等: "整合子介导的大肠埃希菌临床菌株多重耐药研究", 《临床检验杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862327A (en) * | 2015-05-18 | 2015-08-26 | 王冬国 | Novel integron containing multiple drug-resistant gene cassettes |
| CN104878037A (en) * | 2015-05-18 | 2015-09-02 | 王冬国 | Integron In1085 |
| CN105039357A (en) * | 2015-07-09 | 2015-11-11 | 王冬国 | Plasmid fragment carrying novel gene qepA3 |
| CN106047898A (en) * | 2016-05-10 | 2016-10-26 | 王冬国 | Novel integron In1289 containing multiple drug-resistant gene cassettes |
| CN106047900A (en) * | 2016-05-10 | 2016-10-26 | 王冬国 | Novel integron In1287 |
| CN106167802A (en) * | 2016-05-10 | 2016-11-30 | 王冬国 | A kind of novel integron In1290 |
| CN109913399A (en) * | 2019-04-12 | 2019-06-21 | 贵州省水产研究所 | A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application |
| CN109913399B (en) * | 2019-04-12 | 2021-08-20 | 贵州省水产研究所 | Aeromonas intermedia integron containing multiple drug-resistant gene cassettes and obtaining method and application thereof |
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