Affinity purification of plasmid DNA by temperature‐triggered precipitation
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Abstract This report describes a new plasmid DNA purification method, which takes advantage of the DNA‐binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin‐like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water‐soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein‐based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large‐scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines. © 2004 Wiley Periodicals, Inc.Keywords:
Plasmid preparation
Exogenous DNA
The direct extraction of plasmid DNA containing antibiotic resistance genes from complex samples is imperative when studying plasmid-mediated antibiotic resistance from a One Health perspective, in order to obtain a wide representation of all the resistance plasmids present in these microbial communities. There are also relatively few bacterial species from natural environments which can be cultured in vitro. Extracting plasmids from the cultivable fraction of these complex microbiomes may only represent a fraction of the total antibiotic resistance plasmids present. We compared different methods of plasmid extraction from broiler cecal samples, whose resistance could be expressed in a human pathogen-Escherichia coli. We found that kits designed for DNA extraction from complex samples such as soil or feces did not extract intact plasmid DNA. Commercial kits specific for plasmid extraction were also generally unsuccessful, most likely due to the complexity of our sample and intended use of the kits with bacterial culture. An alkaline lysis method specific for plasmid extraction was ineffective, even with further optimization. Transposon-aided capture of plasmids (TRACA) allowed for the acquirement of a small range of resistance plasmids. Multiple displacement amplification provided the broadest range of resistance plasmids by amplifying all extracted circular plasmid DNA, but the results were not reproducible across all samples. Exogenous plasmid isolation enabled the extraction of resistance plasmids from the microbial fraction by relying on the mobility of the plasmids in the sample. This was the most consistent method from which we obtained a range of resistance plasmids from our samples. We therefore recommend the use of the exogenous plasmid isolation method in order to reliably obtain the greatest representation of the total antibiotic resistance plasmidome in complex samples. While this method has limitations, it is one which will vastly increase our current knowledge of antibiotic resistance plasmids present in complex environments and which are capable of transferring to a human and animal pathogen and environmental contaminant.
Human pathogen
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Four therapeutically important strains of Lactobacillus acidophilus designated as R, 301, 1899 and NCFM were screened for the presence of plasmids. Two lysis methods were used for the isolation of plasmid DNA: an alkaline method and a more gentle technique. It was found that the gentle lysis method yielded better plasmid DNA both quantitatively and qualitatively. All four strains studied apparently possess plasmids. The strains 301 and NCFM possessed one plasmid each, with a size of 4.2 kb, whereas R possessed three plasmids (3.5, 2.4 and 2.1 kb) and 1899 possessed two plasmids (4.1 and 4.2 kb). Restriction analysis revealed that the plasmid DNA from strain R was cleaved by Bam HI but not by Hind III and Eco RI. The plasmid DNA from the remaining three strains was cleaved by all three restriction enzymes used.
Alkaline lysis
Plasmid preparation
Strain (injury)
Restriction map
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Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.
Neisseria gonorrhoeae
Plasmid preparation
Alkaline lysis
Restriction map
T-DNA Binary system
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Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama , having molecular weights of 42 × 10 6 and 5.8 × 10 6 , respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm 3 , respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi + or fi − phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl 2 -treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli , the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit 3 H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.
Plasmid preparation
T-DNA Binary system
Kanamycin
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A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.
Replicon
Plasmid preparation
Colicin
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Plasmid preparation
Homology
Southern blot
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참김(P. tenera)의 total DNA를 Hoechst dye/CsCl 농도구배 초고속 원심분리로 분리하였을 때, 두개의 band가 분리되었다. Hoechst dye는 핵산염기서열중 AT가 풍부한 배열에서만 결합하는 염색시약으로서 상부 band는 세포소기관(엽록체, 미토콘드리아) 핵산 및 plasmid 부분이 염색되고, 하부 band는 chromosome의 핵산을 나타내는 부분이다. 이 plasmid는 엽록체나 미토콘드리아와 함께 분리되었으므로 엽록체나 미토콘드리아 유래의 plasmid일 가능성이 매우 높다. 이 plasmid는 다른 홍조류나 북미산의 김종류보다 핵산분리가 힘들고, 또 DNA의 농축이 어려운 한국산 양식김에서(참김)에서 처음으로 분리된 plasmid DNA이었다. 이 plasmid가 선형(linear) conformation인지 구형(circular) conformation인지 확인하기 위해 0.8% agarose 젤+TBE 완충액과 1.2% agarose 젤+TAE(Tris-acetic acid EDTA)전기영동 완충액으로 따로 전기영동해 본 결과 각각의 전기영동 시스템에서 서로 다른 결과를 가져왔다. 0.8% gel에서는 1.8kb의 marker자리로 그리고, 1.2% gel에서는 2.0kb marker의 자리로 영동되었다. 따라서 이 plasmid는 전기영동 조건에 따라 영동거리가 변화함으로써 linear가 아닌, circular plasmid로 확인되었다. 그리고 소량의 김(0.05g)으로 plasmid을 분리한 결과 상당량의 plasmid를 분리할 수 있었던 것으로 보아 본 plasmid는 high copy plasmid로 추산되었다. 이 plasmid의 제한효소 mapping을 하기 위해 agarose gel의 plasmid band부분을 칼로 절단하여 gel elution을 실시한 후에 선택된 여러 가지 제한효소를 처리하여 본 후 unique한 제한효소을 선택하여 절단 반응시켜 본 결과 EcoRI, SacI, SalI의 처리에서는 2.5kb 정도의 크기로 나타났으며, PstI과 BglI을 혼합 처리한 결과에서는 2.2kb 크기로 판정되었다. 따라서 이 plasmid는 최소 2.5kb이상 크기를 가진 고리형 plasmid라 결론지었다(Fig. 4). 이플라스미드를 이용해서 김형질전환벡터작성이 진행중이다.
Plasmid preparation
Tenera
Agarose
Agarose gel electrophoresis
Porphyra
T-DNA Binary system
PstI
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Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.
Plasmid preparation
Wild type
T-DNA Binary system
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A strain of Escherichia coli K-12 carrying eight compatible and distinguishable plasmids was constructed. The amounts of plasmid DNA (measured as supercoiled molecules) per chromosome in this strain was about equal to the sum of the plasmid DNAs, extracted under controlled conditions, from strains each carrying one of the eight plasmids. Analysis of these DNA preparations showed that each plasmid in the multiplasmid strain was present in the same proportion per chromosome as in the single-plasmid strains. Also the level of phenotypic expression of each plasmid in the multiplasmid strain was the same as in the single-plasmid strains. Each plasmid, therefore, appears to control its own copy number irrespective of the presence of other compatible plasmids.
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Strain (injury)
T-DNA Binary system
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Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.
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T-DNA Binary system
Strain (injury)
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Citations (95)