• Journal of Microbiology and Biotechnology
• /
• v.20 no.10
• /
• pp.1463-1470
• /
• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Journal of fish pathology
• /
• v.8 no.1
• /
• pp.31-36
• /
• 1995

Hc nuclear polyhedrosis virus DNA genome was digested with EcoRI endonuclease, these partial fragments were recombined into the pUC8 plasmid vector and transformed in E. coli JM 83 cell. The genome DNA has 24 EcoRI fragments and 12 fragments of them were subcloned. The four recombinants were named as eNP-O, eNP-Q, eNP-R and eNP-S. The expression of foregin gene of the recombinants was investigated by analysing protein patterns on the SDS-PAGE. The eNP-O, eNP-Q and eNP-R were expressed a different weight of protein as comparision with potypeptide bands of E. coli JM 83 host cell.

• Korean Journal of Microbiology
• /
• v.22 no.4
• /
• pp.249-255
• /
• 1984

To develop the host-vector system for industrial Coryneform bacteria that seemed to be the most suitable microorganisms for molecular breeding of genes involved in the production of amion acids, nucleotides, and other products of industrial interest, broad host range E. coli plasmid R 1162 DNA was transformed into Brevibacterium ammoniagenes and the plasmids pKU6 isolated from a transformant was physically characterized. All other plasmids from the transformed cells except pKU6 exsisted as multimeric forms in Brevibacterium ammoniagenes. The plasmid DNA was retransformed into Corynebacterium glutamicum with a high frequency ($1.32{\times}10^{-1}$ per cell) and maintained stably both in Brevibacterium ammoniagenes and Corynebacterium glutamicum after 100 generations of cultures with 25-30 copy number per cell. The size of both plasmid pKU6 and plasmid R1162 were the same and restriction maps by EcoR I, Ava I, Pst I, Pvu II and Hinc II were also similar.

• Korean Journal of Microbiology
• /
• v.26 no.2
• /
• pp.113-121
• /
• 1988

Ozone, an atmospheric pollutant, can damage similar UV and X-rays DNA and its components. It is possible then that the KNA damage produced by this gas are similar, to some extent, to those of radiations and that they could be repaired by the same DNA repair mechanisms. It has been observed in Escherichia coli that radiosensitive strains such as lex A, rec A and pol A, all deficient to some extent for DNA repair, are more sensitive to ozone than a wild type strain. We have thendetermined the ozone resistance and host-cell reactivation of ozone-damaged T3 phages for the E. coli double mutants pol A, lex A, uvr B, lex A, uvr A, rec A and rec A lox A. According to the results, the DNA polymerase 1 plays a key role in ozone resistance and Type 11 mechanism and/or shory patch excision repair are the most important for it. The interactions between the different DNA repair mechanisms are secondary. There is a strong correlation between ozone resistance and the capacity to reactivate T3 phages damaged by ozone.

• Microbiology and Biotechnology Letters
• /
• v.18 no.6
• /
• pp.640-646
• /
• 1990

We have cloned the cdd gene from E. coli C600 using (cdd-) as a host. From the sequenced promoter region of E=, coli cdd gene which has been determined by Valentin-Hansen P. (1985), we synthesized the 23 mer oligonucleotides corresponding to the transcription initiation region and used as a probe for cloning of the cdd gene by Southern blotting. The isolated fragments in the blotting were introduced to the colony hybridization after transforming it into the E. coli JF611 (cdd-, pyr double mutant), and we identified the hybridized band at 27 kb long. From the original insert of 27 kb fragment in theBamHI site of pBR322, the 5.3 kb fragment containing the cdd gene was isolated by subsequent deletion and subeloning. From the derived plasmid pTK509, further deletion and subcloning were performed and clarified that the cdd gene was located in the 2.1 kb of SaZI/DraI segment in the insert of pTK605. The polypeptide encoded by the cloned DNA was appeared to be a molecular mass of 33,000.

• Korean Journal of Microbiology
• /
• v.25 no.1
• /
• pp.57-66
• /
• 1987

Mutants of E. coli which showed increased sensitivity to MMS(methylmethane sulfonate)were isolated by MNNG mutagenesis and characterized by enzymatic assay, survival of simple alkylating agents and host-cell reactivation. E.coli mutant, 5-62, which showed absolute deficiency in 3-methyladenine DNA glycosylase II activity and had low capability of reactivating MMS-treated phage charon 35 was very sensitive to MMS and MNNG. NNS gene which confered resistance to the lethal effects of MMS was cloned in 5-62 strain. 5-62 mutants carrying recombinant plasmid, pMRG 1, which acquired resistance to the lethal effects of MMS had normal sensitivity to MNNG. Resistance to MMS was somewhat increased after they were treated with 0.5.$\mu$g MNNG/ml for 2 hours at $37^{\circ}C$. Although recombinant plasmid, pMRG 1, did not complement alk A mutation in 5-62 and ada mutation in 1-27 mutnat, mutnats transformed with this plasmid showed more capability of reactivating MMS treated phage than mutants.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.1
• /
• pp.30-38
• /
• 2010

The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions that enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e., M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system, we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild-type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness.

• KSBB Journal
• /
• v.11 no.1
• /
• pp.37-45
• /
• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.2
• /
• pp.403-411
• /
• 2005

The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.

• Fisheries and Aquatic Sciences
• /
• v.12 no.1
• /
• pp.24-28
• /
• 2009

A tetracycline resistant Vibrio parahaemolyticus, capable of growing on TCBS medium containing tetracycline, was isolated from cultivated fishes. A gene responsible for the tetracycline resistance was cloned from chromosomal DNA of the V. parahaemolyticus strain using Escherichia coli KAM3, which lacks major multi-drug efflux pumps (${\Delta}acrB$) as host cells. The nucleotide sequence and homology analysis revealed an open reading frame (ORF) for tetracycline resistance protein (TetB). In order to characterize the antibiotic resistance of TetB originated from the V. parahaemolyticus strain, the gene was sub cloned into plasmid pSTV28. The resulting plasmid was designated as pSTVTetB and transformated into E. coli KAM3. E. coli KAM3 cells harboring the recombinant plasmid pSTVTetB are able to grow on plates containing tetracycline and oxytetracycline but not doxycycline, indicating that the tetB gene confers the tetracycline- and oxytetracycline-resistance to the host cell.