• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1630-1633
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• 2008

A novel putative toxin-antitoxin segregational stability system named KyAB system was identified in a novel native plasmid pBMB8240 from Bacillus thuringiensis strain YBT-1520, based on sequences homology with other toxin-antitoxin systems, the lethal activity of the KyB putative toxin in Escherichia coli and the stabilizing effect of the kyAB system in Bacillus thuringiensis. Secondarily, the native plasmid pBMB9741 from the same strain was resequenced and the corrected plasmid was named as pBMB7635. Based on sequence homology with the tasAB system and the lethal activity of toxin protein in Escherichia coli, a tasAB-like putative toxin-antitoxin system was identified on pBMB7635.

• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.168-173
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• 1993

The expression kinetics of human lipocortin I (LCI), a potential anti-inflammatory agent, was studied in the shake-flask and fermenter cultures of Saccharomyces cerevisiae carrying a galactose-inducible expression system. The cell growth, expression level of LCI, and the plasmid stability were investigted under various galactose induction conditions. The expression of LCI was repressed by the presence of a very small amount of dextrose in the culture medium, but it was induced by galactose after dextrose became completely depleted. The optimal ratio of dextrose to galactose for lipocortin I production was found to be 1.0 (10 g/l dextrose and 10 g/l galactose). With optimal D/G ratio of 1.0 and the addition of galactose prior to dextrose depletion, LCI of about 100~130 mg/l was produced. LCI at a concentration of 174 mg/l was porduced in the fed-batch culture, which was nearly a twice as much of that produced in the batch culture. The plasmid stability was very high in all culture cases, and thus was considered to be not an important parameter in the expression of LCI.

• BMB Reports
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• 제53권8호
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• pp.442-447
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• 2020

The non-viral delivery of genes into macrophages, known as hard-to-transfect cells, is a challenge. In this study, the microporation of a CpG-free and small plasmid (pCGfd-GFP) showed high transfection efficiency, sustainable transgene expression, and good cell viability in the transfections of Raw 264.7 and primary bone marrow-derived macrophages. The non-viral method using the pCGfd vector encoding anti-EGFR single-chain Fv fused with Fc (scFv-Fc) generated the macrophages secreting anti-EGFR scFv-Fc. These macrophages effectively phagocytized tumor cells expressing EGFR through the antibody-dependent mechanism, as was proved by experiments using EGFR-knockout tumor cells. Finally, peri-tumoral injections of anti-EGFR scFv-Fc-secreting macrophages were shown to inhibit tumor growth in the xenograft mouse model.

• 대한수의학회지
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• 제34권4호
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• pp.759-768
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• 1994

E coli strains isolated from pigs were investigated with respect to antimicrobial resistances and prevalence of R-plasmids. Also determined were properties of R-plasmids by plasmid conjugation, curing and southern hybridization using gene probes. All of 400 E coli strains were resistant to CL and SU, and 0.3% to 96.8% of the strains were resistant to most antimicrobials such as TC, PG, AM, SM, CP, GM, EM, NM, etc, while all strains were sensitive to AK. All strains were also multiply resistant to three to twelve antimicrobials. The resistances to PG, SM, TC, AM, CP, SU and ST were transferable and supposed to be mediated by R-plasmids which were opportunistic for transposition into chromosome. Plasmids bigger in size than chromosomal DNA were considered as R-plasmids and most plasmids in small size (<4Kb) proved as cryptic plasmids or nonconjugative R-plasmids. In a strain(No 99), AM resistant property was determined from both chromosomal DNA and R-plasmid DNA which is bigger in size than chromosome.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• BMB Reports
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• 제30권2호
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• pp.162-165
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• 1997

RNA I. a negative controller of ColE1-type plasmid replication, is metabolized by several RNases in Escherichia coli. Two small derivatives of RNA I are accumulated at nonpermissive temperatures in an E. coli strain carrying the rnpA49 mutation, a thermosensitive mutation in the rnpA gene encoding the protein component of RNase P. A primer extension analysis was carried out to compare 5' processing of RNA I in the E. coli rnpA49 cells at both permissive and nonpermissive temperatures. Derivatives of RNA I having different 5' ends were observed in the cells grown at permissive and nonpermissive temperatures. Some of the derivatives may be generated by the cleavage of RNase P.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.278-284
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• 1994

Streptomyces spp. purchased from American Type Culture Collection and Institute for Fermentation in Osaka, and donated from Northem Regional Research Laboratory, and those isolated from soil samples were assayed to isolate many plasmids harboring streptomycetes. Among these qrganisms, 5 small size-plasmid carrying organisms SNUS 8810-597A, 8810-600, 8810-754, 8811-344, and 8811-347 were characterized and their plasmids pSJ597, pSJ600, pSJ754, pSJ344, and pSJ347 were isolated in a large scale. The plasmid harboring organisms were sensitive to neomycin, kanamycin, gentamicin, and thiostreptone, but some of them showed weak or strong resistance against streptomycin, chloramphenicol, ampicillin, and tetracycline. It was confirmed that pSJ597 and pSJ600 do not carry antibiotic biosynthetic genes. pSJ600 showed a pock-forming character.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2001년도 10주년 기념 국제학술 심포지움 및 추계학술대회
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• pp.82-82
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• 2001

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Journal of Plant Biology
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• 제32권1호
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• pp.33-40
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• 1989

Polysomal polyadenylated mRNAs which were purified from pea leaves were fractionated by sucrose grandient sedimentation. Fractions corresponding to the peak at 11.5S were found to contain mostly mRNA encoding the precursor polypeptide to the small subunit of ribulose bisphosphate carboxylase (rbcS) by in vitro translation in wheat germ extract. Double-stranded cDNA which was synthesized from the 11.5S mRNA was cloned into Hind III site of plasmid pBR 325. A cDNA clone, H24, was identified to code for rbcS. In vitro translation product of the hybridization-selected mRNA was molecular weight 20,000, presumably the precursor of rbcS. The nucleotide sequences of the H24 showed almost complete homology with the sequences encoding the transit peptide of the rbcS-3A gene which was reported by Fluhr et al.(1986).