• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1123-1126
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• 2011

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.

• Archives of Pharmacal Research
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• 제18권1호
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• pp.31-37
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• 1995

S. pneumoniae (pneumococcus) gene cloning and library construction in E. coli multicopy plasmid has been hampered, in part, by instability problems. In this study, stability of pneumococcus gene library in cosmid vector and pACYC184 was examined. Pneumococcus library in the cosmid vector pHC79 was extermely unstable that most of the recobinant clones were degenerated rapidly. Only 2 out 849 clones were stable and had appropriate insert size. Pneumococcus library in pACYC184 was also so unstable that the pneumococcal inserg and/or part of the vector were deleted. However, the instability problems seemed to be resolved when transcription teminator plasmid was employed for pneumococcus library construction.

• Applied Biological Chemistry
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• 제31권3호
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• pp.219-225
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• 1988

Avian Sarcoma Virus의 plasmid DNA중(中)의 역 이사효소의 유전자(遺傳子)를 온도의존성(溫度依存性) 발현(發現) vector인 pPL-lambda에 cloning하여 온도(溫度)에 민감한 phage ${\lambda}$의 repressor인 cI857 gene을 갖고 있는 bacteriophage lysogen인 N4830에 transformation시켰다. transformant를 pL promoter의 발현(發現)을 억제(抑制)하는 저온(低溫)$(28^{\circ}C)$에서 배양(培養)시킨 뒤, 이 repressor를 억제(抑制)하여 transcription을 촉진(促進)하게 하는 고온(高溫)$(42^{\circ}C)$에서 배양(培養)시킨 다음 균체(菌體)를 회수(回收)하여 RNA를 추출(抽出)하고 분석(分析)을 한 결과(結果) 도입(導入)된 역전사 효소 유전자(遺傳子)의 전사(轉寫)가 고온(高溫)에서 증대(增大)되었다.

• Preventive Nutrition and Food Science
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• 제9권4호
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• pp.324-329
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• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• 미생물학회지
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• 제27권2호
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• pp.92-97
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• 1989

the vector, pGUR19, for Rhizobium gene manipulation, was constructed by combining the replication and mobilization function of indigenous multicopy plasmid from Acacia(Robinia pseudoacacia L.) Rhizobia sp86 with E. coli cloning vehicle, pBR322. The vector could be efficiently mobilized by RP4 tra function incorporated into chromosome of E. coli named SM10 and efficiently transferred to various gram negative hosts including Rhizobium and Afrobacterium by transformation. Mobilization frequency of the constructed vector was ranged from $1.2\times 10^{-2}$ (E.coli HB 101) to $4.6\times 10^{-4}$ (A. tumefaciens 15955) and transformation frequency was ranged from $5.4\times 10^{-7}$(E. coli HB101) to $1.2\times 10^{-10}$ (A. tumefaciens 15955). The vector, pGUR19, was stably replicated and maintained in a variety of Rhizobium and Agrobacterium.

• Journal of Microbiology
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• 제44권5호
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• pp.515-522
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• 2006

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

• KSBB Journal
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• 제11권1호
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• pp.115-123
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• 1996

zooglan생합성에 필수적인 gene cluster를 clone하기 위해 2종류의 변이주가 분리되었다. Zoogloea ramigera 115는 협막형 다당(주로 zooglan)을 생산한다. 115 균주로 접합시키고 생산물을 용이하게 분리하기 위하여 반복된 원심분리와 선별을 통해 협막을 만들지 않는 slime형 생산균주를 분리하였다. 세포외 다당 생산 능력이 결여된 변이주를 전통적인 transposon(Tn5) 기술을 사용하여 얻었고 달라진 colony 형태와 celluflour결합 성질에 의해 선별하였다. 이들 변이주들은 범용숙주범위 cosmid vector안에 건설된 Z.ramigera 115slime gene library 와 helper plasmid로의 3양친 접합에 의해 보상되었다.

• Applied Biological Chemistry
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• 제31권3호
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• pp.267-273
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• 1988

Transformed, hybrid strains of the yeast Saccharomyces capable of simultaneous secretion of both glucoamylase and ${\alpha}-amylase$ have been produced. These strains can carry out direct, one-step assimilation of starch with conversion efficiency greater than 93% during a 5 day growth period. One of the transformants converts 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains results from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid(pMS12) containing mouse salivary ${\alpha}-amylase$ cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast $2{\mu}$ plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths is glucose, indicating that ${\alpha}-amylase$ and glucoamylase act cooperatively.

• Archives of Pharmacal Research
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• 제27권7호
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• pp.776-780
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• 2004

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFN$\{alpha}$-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the HindIII and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

• 한국환경과학회:학술대회논문집
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• 한국환경과학회 2002년도 봄 학술발표대회 발표논문집
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• pp.445-447
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• 2002

본 연구는 환경친화적인 biosurfactant를 생산하는 Pseudomonas aeruginosa EMS1 and KH7를 rhamnolipid의 rhlR, rhlA, rhlB를 기초로한 primer를 이용하여 752bp, 802pb, 1280bp pcr을 수행하였으며 $pGEM^{(R)}$ / - T Easy Vector gene cloning 하여 Pseudomonas aeruginosa EMS1 and KH7의 Partial Sequencing를 서로 비교하였다. 이들 실험을 통하여 Pseudomonas aeruginosa의 유전적 구조 및 특성을 비교하여 유전적 조작을 위한 기초적인 자료가 되도록 한다.