• Journal of Microbiology and Biotechnology
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• 제7권4호
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• pp.223-228
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• 1997

A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1185-1191
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• 2006

The expression of a pullulanase gene in Pichia pastoris was investigated. The gene encoding pullulanase was cloned by PCR using the chromosomal DNA of Bacillus naganoensis as the template. The expression vector pPIC9K-Pu was constructed by inserting the pullulanase gene into plasmid pPIC9K and then transformed into Pichia pastoris SMD 1168 by electroporation. Activity determination, SDS-PAGE, and PCR amplification indicated that the gene of the pullulanase from B. naganoensis had successfully been expressed in SMD 1168 and the molecular size of the expressed recombinant product was about 119.9 kDa. This is the first report on the successful expression of the pullulanase from B. naganoensis in P. pastoris. The transformant secreted recombinant pullulanase with the activity of 350.8 IU/ml in shake-flask culture. The properties of the recombinant pullulanase were characterized.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.986-988
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• 2002

A plasmid expression vector of pEStxl encoding a mature form of the B chain of the Shiga toxin was constructed without a signal peptide under the control of an inducible n promoter. The encoded protein was purified to 90% by simple heat treatment, and then further purified to 95% by Phenyl-Sepharose and DEAE-Sepharose chromatographies, all in a single day. Accordingly, this expression system and heat treatment could facilitate the rapid purification of gram-scale amounts of the Shiga toxin B subunit from recombinant Escherichia coli cells.

• 한국미생물·생명공학회지
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• 제24권6호
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• pp.657-663
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• 1996

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.277.2-278
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• 2002

Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of simple synthetic vector systems such as cationic lipid- and polymer-complexed plasmid DNA were shown to have a significant transfection activity in vitro but their use in vivo has been hampered by the decrease in transfection efficiency mediated by non-specific electrostatic interactions with serum components. (omitted)

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.352-352
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• 2006

Corynebacterium glutamicum, which is well known as an amino acid fermentation bacterium, has been used as a producer of poly(3-hydroxybutyrate) [P(3HB)]. P(3HB) was synthesized in recombinant C. glutamicum harboring the expression plasmid vector with a strong promoter for cell surface protein gene derived from C. glutamicum and P(3HB) biosynthetic gene operon derived from Ralstonia eutropha. The expression of P(3HB) synthase gene was detected by enzyme activity assay. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5 % (w/w) with molecular weight of $2.1{\times}10^{5}$ and polydispersity of 1.63.

• 산업기술연구
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• 제29권B호
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• pp.115-119
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• 2009

Green fluorescent protein (GFPuv) was displayed on the surface of ethanol-producing bacteria Zymomonas mobilis using N-terminal domain of ice nucleation protein (INP) as an anchoring motif. To evaluate the ice nucleation protein as plausible anchor motif in Z. mobilis, GFPuv gene was subcloned into Zymomonas expression vector yielding pBBR1MCS-3/pPDC/INPN/GFPuv plasmid., INP-GFPuv fusion protein was expressed in Z. mobilis and its fluorescence was verified by confocal microscopy. The successful display of GFPuv on Zymomonas mobilis suggest that INP anchor motif could be used for future fusion partner in Z. mobilis strain improvement.

• 생명과학회지
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• 제13권5호
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• pp.712-717
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• 2003

박테리오신의 일종인 leucocin A를 생산하는 효모의 제작을 위하여 117 bp 길이의 개시코돈과 종지코돈을 포함하는 leucocin A 유전자를 합성하여 효모운반체인 pAUR123에 클로닝하였다. 형질전환된 효모는 부패균의 일종인 고초균(Bacillus subtilis)에 대해 항균활성을 나타냈다. 형질전환 효모로부터 분리한 플라스미드를 주형으로 하고 leucocin A에 특이적인 primer들을 이용하여 PCR 반응을 행한 결과, 효모에 도입된 leucocin A 유전자를 확인할 수 있었다. 이 연구의 결과로 부패하기 쉬운 식품들의 보존성을 향상시키거나, 내성이 생긴 병원균의 생육을 저해하기 위한 항생제로 사용할 수 있는 박테리오신을 산업적으로 생산할 수 있는 효모세포를 제작하였다.

• Archives of Pharmacal Research
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• 제15권1호
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• pp.58-61
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• 1992

Bacillus anthracis 590 having an inducibla resistance determinant to MLS antibiotics was isolated from a soli sample in Korea. The resistance gene (ernJ) was cloned by Southern blotting of chromosomal DNA fragment digested by various restriction enzymes and coloy hybridization method and the cloned plasmid was named as pBA423. The size of inserted DNA fragment of pBS42 vector was about 2.9 kb and the DNA sequence of the subcloned fragment (Hinc II-Hinc II, 1.4kb) WAS determined. The DNA sequence of ernJ was composed of 357 bp for leader region and 861 bp for the structural gene. Because the leader sequence of ernJ was homologous to that of ermK, the expression of ernJ is also thought to be controlled by a transcriptionl attenuation mechanism.

• 미생물과산업
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• 제16권1호
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• pp.18-20
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• 1990

현재 전세계적으로 10,000여개 이상의 실험실에서 생물공학적인 연구가 진행중에 있고, 200여개 이상의 회사에서 생물공학을 이용한 제품이 만들어지고 있다.(Saftlas, 1984). 이와같은 제품으로 생물농약(예, 모기억제에 사용되는 Bacillus thuringiensis var. israelensis 등)이라든가 insulin, 성장호르몬, lymphotoxin및 항암제등의 의약품(Saftlas, 1984)과 식물품종개량제(예, 질소 공정의 vector로 사용되는 Agrobacterium tumerfaciens의 Ti Plasmid)(McDanial, 1981 ; Shaw, 1986) 등이 있다. 그러나 최근 미생물 생태분야에서는 이와같이 만들어진 미생물들 (genetically engineered microorganisms : GEMs)이 자연 생태계에 유출되었을때 야기될 가능성이 있는 biohazaed에 대하여 관심이 집중되고 있다. (Curtiss, 1976 ; Sharples, 1983 ; Rissler, 1984). 토양환경에서 GEM이 유전물질을 전달항 수 있는 기작은 크게 conjugation, transduction, transformation 등이 알려지고 있다. 여기서는 주로 토양환경에서 transformation에 의한 유전물질의 전달과정에 대한 최근 연구동향을 간단히 기술하고자 한다.