• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.209-212
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• 1986

$\alpha$-Amylase gene of Bacillus amyloliquetaciens was cloned on plasmid YEp13, S. cerevisiae-E. coli shuttle vector. Hybrid plasmid pTG17, carrying $\alpha$-amylase gene of B. amyloliquefaciens, was transformed to E. coli and the expression of it in yeast was investigated. This plasmid was unstable in E. coli and produced two minor plasmids, pTG17-1 and PTG17-2, which resulted from the segregation of it. Transformant of S. cerevisiae MC16 with pTG17-1 plasmid was not appeared on SD medium because of the Leu2 gene defection. S. cerevisiae could be transformed by the hybrid plasmid, and $\alpha$-amylase activity of the yeast transformant was detected by somogyi-Nelson method and agar diffusion method.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• Journal of Microbiology
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• v.38 no.2
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• pp.113-116
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• 2000

Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find and additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The results indicates that this system is a promising one, capable of selecting an interacting component.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.819-822
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• 2003

Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based minimal polyketide synthase (PKS) expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orfl, actI-orf2, actI-orf3, actIII, actⅦ, and actIV) from Streptomyces coelicolor, which specify the construction of an orange-fluorescent anthraquinone product aloesaponarin II, a type II polyketide compound derived from one acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in S. parvulus to generate a hybrid type II polyketide compound via gene-specific replacement. The act ${\beta}-ketoacyl$ synthase unit (actI-orfl and actI-orf2) in the expression cassette was specifically replaced with oxytetracycline ${\beta}-ketoacyl$ synthase otcY-orfl and otcY-orf2). This plasmid-based hybrid PKS cassette generated a novel orange-fluorescent compound structurally different from aloesaponarin II in both S. lividans and S. parvulus. In addition, several additional distinctive blue-fluorescent compounds were detected, when this hybrid PKS cassette was expressed in S. coelicolor B78 (actI-orf2 mutant), implying that the expression of plasmid-based hybrid PKS cassette in Streptomyces species should be an efficient way of generating hybrid type II polyketide compounds.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.31-36
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• 1991

A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

• Microbiology and Biotechnology Letters
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• v.27 no.5
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• pp.364-369
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• 1999

In order to obtain the suitable plasmids for constructing plasmid vectors of Bacillus species, endogeneous plasmid DNAs were screended from thermo-tolerant soil bacteria. Based on agarose gel electrophoresis patterns of the isolated plasmid DNAs, two strains harboring small-size plasmids were selected. The isolated were identified to belong to the genus Bacillus on the basis of their morphological and biochemical properties, and named Bacillus sp. 3-3 and 77-8, respectively. The restriction endonuclease maps were determined for four plasmids including two plasmids from each Bacillus isolates. It is interesting that Bacillus sp. 3-3 and 77-8 have an identical plasmid according to the restriction maps. The three kinds of hybrid plasmids constructed by introducing each plasmid of two isolates into a Escherichia coli plasmid vector. pUCCm18 containing chloramplenicol resistance gene active in Bacillus strains, could be replicated in B. subtilis and B. licheniformis. These plasmids are very stable in B. subtilis, suggesting that the Bacillus plasmids identified in this work would be useful for development of new cloning vectors for Bacillus strains.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.226-229
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• 1991

In our laboratory a R plasmid pKU10 was isolated from Pseudomonas and its characteristics were investigated. In this study, as a basic work to improve its utility as a cloning vehicle, restriction patterns of pKU10 were analyzed for other various restriction enzymes in addition to restriction evdonucleases previously examined. As a result, pKU10 DNA has two cleavage sites for ClaI and HpaI, and three sites for AvaI. The restriction map of pKU10 was supplemented with AvaI, ClaI, and HpaI. From the result of this experiment, the usefulness of PKU10 as a cloning vector in Pseudomonas will be enhanced by constructions of mini-plasmid or hybrid plasmids.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.236-242
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• 1990

To develop the new strains of microorganisms having the degradative ability for various aromatic hydrocarbons, the hybrid plasmid pKG2 having the 2,4-Dichlorophenoxyacetic acid(2,4-D) degradative genes, the hybrid plasmid pKG3 containg the naphthalene degradative genes and TOL plasmid were introduced into Pseudomonas putida KUD 12 and P. putida KUP 10 by transformation or conjugation which originally have the degradative ability of the synthetic surfactants and phthalate esters, respectively. From P. putida KUD12, the new strains of P. putida KUD101(pKG2), KUD102(pKG3), KUD103(TOL), and KUD202(pKG3, TOL) were obtained, and KUD106(pKG2), KUD107(pKG3), KUD108(TOL) were originated from the P.putida KUP10. The degradative abilities in P. putida KUD101, KUD102 and KUD107 were similar with those of the original strains. The P. putida KUD103, KUD106 and KUD202 had a little lower and P. putida KUD108 had a better degradative abilitie than those of the original ones. In the case of mixed cultures, the mixed culture of KUD107 and KUD108 had a better degradative abilities than those of the other mixed cultures.

• 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.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.519.2-519
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• 1986

고온 호알카리성 Bacillus K-17의 xylanase유전자의 구조해명과 대량 생산 균주를 개발하기 위채 Bacillus K-17의 염색체를 pER 322를 사용하여 E. coli에 형질전환시켜 xylanase 활성을 나타내는 형질전환체를 얻었다. 이 형질전환체에서 hybrid plasmid를 분리하여 제한효소로 mapping하였고 이 유전자가 Bacillus K-17유래인가를 hybridization에 의해 확인하였다. Recombinant plasmid pAX 1113은 5.1kb HindIII 절편을 가졌으며 BgIII site가 두곳, ECoRI과 pst site가 한곳이었으며 효소를 정제한 결과 Bacillus K-17이 생산하는 두 가지 xylanase중에서 xylanase I과 동일하였다.