• Microbiology and Biotechnology Letters
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• v.15 no.5
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• pp.319-327
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• 1987

In order to develop useful plasmid vectors for Zymomonas cells, attempts were made to isolate natural plasmids from Z. mobilis ATCC10988. Among a few plasmids isolated, a small plasmid of 3.9 Kb size was chosen and designated as pZM3. By introducing the replication origin of pZM3 into pBR325, a hybrid plasmid vector of 8.4 Kb size, pHZ22, was constructed. This vector contained chloramphenicol resistant gene as a selectable marker and proved to be conjugally transmissible and stably maintained in Z. mobilis. Tetracycline resistant gene was isolated from RP4 and introduced into pHZ22 to make a new vector called pHZT224 of 10.7 Kb size. Through n series of experiments, it was evident that these plasmid vectors containing selectable markers of chloramphenicol and tetracycline resistance were shuttle vectors functional in Z. mobilis as well as E. coli.

• Korean Journal of Veterinary Service
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• v.23 no.1
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• pp.77-91
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• 2000

This study was conducted to investigate the antibiotic resistance and plasmid profiles of 58 salmonella spp isolated from mesenteric lymphnodes of slaughter pigs in Kyoungbuk province during the period from September 1997 to June 1998. The results obtained are as follow that all isolates were susceptible to amikacin, gentamicin, ciprofloxacin and enrofloxacin, and the majority of isolates were highly susceptible to norfloxacin, colistin, nalidixic acid and apramycin while they were moderately susceptible to kanamycin, cephalothin, chloramphenicol, ampicillin, trimethoprim and penicillin. The majority of isolates were over 90% resistant rates to lincomycin, sulfadimethoxine, vancomycin, methicillin and erythromycin. The plasmid profiles of 58 salmonella spp are developed 1 to 4 fractions, 0.9 to 29.5 Kb molecular range sizes and U strains (45.5%) were showed plasmid profiles by agarose gel electrophoresis. 5 derby harbored 29.5 Kb and 7 Kb, and S schwarzengrund had 14 Kb and 0.9 Kb harboring sizes. Four of 10 S agona and 2 of 4 S typhimurium were harbored 3.1 Kb and n.5 Kb, respectively. Thirty-five untypable strains are developed variable size fractions its showed small size plasmid profile less than 6 Kb and 22 (62.8%) of them had no detectable plasmids.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.47-52
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• 1986

A heat-labile enterotoxin and no heat-stable enterotoxin producing($LT^+ST^-$) plasmid (110 kilobases in size) was isolated from an enterotoxigenic Escherichia coli of human strain O15:H11 and used for analysis of the $LT^+$ deoxyrionucleic acid region using recombinant DNA technology. A DNA segment containing the $LT^+$ DNA region which was one restriction endonuclease BamHl fragment(6.2 kb in size) was joind to a small multicopy plasmid, pUC9. E. coli K-12 strain, JM103 harboring the chimeric plasmid produced greater amounts of LT than did the enterotoxigenic E. coli O15:H11 strain. The BamHl fragment was further digested with various restriction endonucleases and contained no HindIll restriction site which is an essential in $LT^+ST^+$ plasmid. The detailed DNA sequencing of this $LT^+ST^-$ plasmid is required.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.217-223
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• 1993

To investigate the nature and location of the nisin-resistance determinant of Lactococcus lactis subsp. lactis 7962 (L. lactis 7962), a total plasmid DNA prepared from L. lactis 7962, a nisin producer, was used to transform L. lactis subsp. lactis LM0230, a plasmid-free and nisin-sensitive strain, by protoplast mediated transformation procedures. All of the nisin-resistant transformants acquired the ability to utilize sucrose at the same time, confirming the close linkage between these two determinants in L. lactis 7962. The plasmid DNA profiles of a few selected nisin-resistant transformants were examined by agarose gel electrophoresis. No common plasmid was found among the transformants and some small plasmids previously not present in L. lactis 7962 were detected. These transformants were named as L. lactis KL1, KL2, KL3, KL4, or KL5, respectively based on their plasmid profiles. Growth curves of all transformants were similar to that of L. lactis LM0230, but different from that of L. lactis 7962. L. lactis KL5 showed the highest level of resistance to nisin, growing up to 1, 200 IU nisin/ml after 40 hr incubation. Some nisin-sensitive derivatives of KL1 or KL2 were obtained by plasmid curing experiments. The plasmid DNA profiles of the nisin-sensitive KL1 derivatives were apparently the same as that of the KL1. All of the nisin-sensitive KL2 derivatives were plasmid-free, but a nisin-resistant strain with no apparent plasmid was also obtained. These results indicate that the nisin-resistance of the $Nis^r$ transformants is presumably mediated by the chromosomally located gene(s) rather than plasmid-encoded gene(s).

• BMB Reports
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• v.37 no.3
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• pp.351-355
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• 2004

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a singe tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1408-1414
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• 2009

The obtention of high yields of purified plasmid DNA is viewed as an essential issue to be considered towards efficient production of DNA vaccines and therapeutic plasmids. In this work, Escherichia coli $DH5\alpha$. bearing the pVAXI-LacZ plasmid was grown in a developed semi-defined medium at different temperatures and tryptone concentrations. Analysis of pDNA yields and E. coli morphology revealed that at higher temperatures (37 and $40^{\circ}C$), higher specific yields and E. coli filamentation were obtained. However, the best results were achieved when a lower tryptone concentration was used. This approach was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure, and favoring the attainment of quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA amplification was improved and a specific yield of 20.43 mg pDNA/g dcw was achieved, proving that this strategy can improve pDNA yield even at a small scale.

• Biomedical Science Letters
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• v.9 no.2
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.292-294
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• 1996

To investigate the relationship between two tetracycline resistance ($Tc^r$) plasmids, the 24.82-kb pKHl and the 4.44-kb pKH6, of Staphylococcus aureus isolated in Korea, cloning of the 4570-bp HindIII fragment into pBluescript II $KS^+$ after partial digestion of the $Tc^r$ plasmid pKHl with HindIII and sequence determination of that fragment were carried out. Analysis of the sequences revealed that the 4570-bp HindIII fragment contained a 4011-bp fragment of the small $Tc^r$ plasmid pKH6 flanked by the partial sequences of IS431mec. It was concluded from the above result that the pKHl was produced by integration of the partial sequence of the pKH6 into another plasmid via IS431mec.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.725-729
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• 2016

A novel genome-engineering tool in Clostridium acetobutylicum was developed based on single-crossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.837-842
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• 2013

A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.