• Title/Summary/Keyword: Shuttle vector

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Expression of the Bacillus stearothermophilus NO2 CGTase gene in Saccharomyces cerevisiae (Saccharomyces cerevisiae 내에서 Bacillus stearothermophilus NO2 CGTnse 유전자의 발현)

  • 유동주;박현이;전숭종;권현주;남수완;김병우
    • Microbiology and Biotechnology Letters
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    • v.30 no.3
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    • pp.206-209
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    • 2002
  • For the expression of CGTase gene(cgtS) kom Bacillus stearothemophilus NO2 in Saccharomyces cerevisiae, cgtS gene was subcloned into the Eschepichia coll-yeast shuttle vector, pVT103-U. The constructed plasmid, pVT-CGTS was introduced to 5. cemi-siae 2805 cell, and then the cgtS gene under the control of adhl promoter was successfully expressed in the yeast transformant and 87% of the total activity was detected into the fermentation medium. Therefore, the signal peptide of B. stearothemephilus NO2 CeTase showed high secretion efficiency in 5. cerevisiae. Optimal conditions of the recombinant yeast cell f3r expression of CGTase was achieved, when 5. cerevisiae 2805/pv7-CGTS was cultivated on YP medium at 2% dextrose, pH 5.5,$30^{\circ}C$ and the expression level of CGTase was 0.624units/mL for 48 h culture.

Construction of a Corynebacteriurn glutarnicum-Escherichicr coli Shuttle Vector and Cloning the Homoserine ehydrogenase Gene from C. glutamicum (Corynebacterium glutamicum-Escherichia coli Shuttle Vector 개발과 C.glutamicum 의 Homoserine Dehydrogenase Gene Cloning)

  • 최신건;박종현;신현경
    • 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.

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Construction of a Novel Shuttle Vector for Tetragenococcus species based on a Cryptic Plasmid from Tetragenococcus halophilus

  • Min Jae Kim;Tae Jin Kim;Yun Ji Kang;Ji Yeon Yoo;Jeong Hwan Kim
    • Journal of Microbiology and Biotechnology
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    • v.33 no.2
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    • pp.211-218
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    • 2023
  • A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Emr), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

Molecular Cloning of the Gene Coding for 3-Isopropylmalate Dehydrogenase of Kluyveromyces fragilis (Kluyveromyces fragilis의 LEU gene의 Cloning)

  • 박성희;이동선;우주형;김종국;홍순덕
    • Microbiology and Biotechnology Letters
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    • v.18 no.3
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    • pp.305-308
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    • 1990
  • In order to clone the gene coding for 3-isopropylmalate dehydrogenase of Muyveromyces fragilis, a shuttle plasmid vector pHNll4 was used. It can serve as a cloning vector in Saccharomyces cerevisiae DBY746 for other Sau3AI-cleaved DNA segment of Kluyveromyces fragilis. Two cloned fragments which complement the leu2 mutation of Saccharomyces cerevisiae and E, coli were obtained. Their length was 4.4 kb an 3.5 kb, and their orientation was opposite each other. From the fact that the two recombinant plasmids were expressed in Saccharomyces cerevisiae and E, coli, probably the two inserts had the promoter of Ktuyveromyces fi-agilis and that of Kluyveromyces fiagilis was efficiently assosiated with RNA polymerase of Saccharomyces cerevisiae and E. coli. According to the result of Southern hybridization, we thought that the cloned fragment has low homology with 3-isopropylmalate dehydrogenase coding region of E. coli and Saccharomyces cerevisiae.

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Development of Leuconostoc sp. Host Vector System

  • Eom, Hyun-Ju;Park, Myeong-Soo;Ji, Geun-Eog;Han, Nam-Soo
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 2004.06a
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    • pp.323-327
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    • 2004
  • Leuconostoc citreum CBUE isolated from kimchi proved to harbor a small cryptic plasmid, pNS75. The complete nucleotide sequence of pNS75 was 1,821 bp and had a low G+C content of 39.2%. Computer analysis using DNASIS revealed one open reading frame (ORF), having ATG as putatitive start condon and potentially encoding proteins with molecular mass of 38 kDa. The chimeric plasmid pLeuCM was first constructed wih pNS75, pUC19 and chroamphenicol acetyltransferase (CAT) from Staphylococcus sp.. pLeuCM replicated and expressed chroamphenicol acetyltransferase in Leuconostoc citerum CBNF after transformation. To test the availability of shuttle vector as cloning vehicle of foreign gene, $\alpha$-amylase gene of Streptococcus bovis was cloned and all transformants secreated the $\alpha$-amylase successfully. The result indicates that pLeuCM is a potential shuttle vector for Leuconostoc spp. and lactic acid bacteria.

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Construction of a Lactococcal Shuttle/Expression Vector Containing a $\beta$-Galactosidase Gene as a Screening Marker (선별마커로써 $\beta$-Galactosidase 유전자를 포함한 Lactococcus용 셔틀/발현 벡터 제조)

  • Han Tae Un;Jeong Do-Won;Cho San Ho;Lee Jong-Hoon;Chung Dae Kyun;Lee Hyong Joo
    • Microbiology and Biotechnology Letters
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    • v.33 no.4
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    • pp.241-247
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    • 2005
  • A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

Methods for Swing Recognition and Shuttle Cock's Trajectory Calculation in a Tangible Badminton Game (체감형 배드민턴 게임을 위한 스윙 인식과 셔틀콕 궤적 계산 방법)

  • Kim, Sangchul
    • Journal of Korea Game Society
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    • v.14 no.2
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    • pp.67-76
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    • 2014
  • Recently there have been many interests on tangible sport games that can recognize the motions of players. In this paper, we propose essential technologies required for tangible games, which are methods for swing motion recognition and the calculation of shuttle cock's trajectory. When a user carries out a badminton swing while holding a smartphone with his hand, the motion signal generated by smartphone-embedded acceleration sensors is transformed into a feature vector through a Daubechies filter, and then its swing type is recognized using a k-NN based method. The method for swing motion presented herein provides an advantage in a way that a player can enjoy tangible games without purchasing a commercial motion controller. Since a badminton shuttle cock has a particular flight trajectory due to the nature of its shape, it is not easy to calculate the trajectory of the shuttle cock using simple physics rules about force and velocity. In this paper, we propose a method for calculating the flight trajectory of a badminton shuttle cock in which the wind effect is considered.

Characterization of Plasmid pKJ36 from Bifidobacterium longum and Construction of an E. coli-Bifidobacterium Shuttle Vector

  • Park, Nyeong-Soo;Shin, Dong-Woo;Lee, Ke-Ho;Ji, Geun-Eog
    • Journal of Microbiology and Biotechnology
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    • v.10 no.3
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    • pp.312-320
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    • 2000
  • Abstract The full sequence of the plasmid pKJ36, which was derived from Bifidobacterium longum KJ, was determined and analyzed to construct shuttle vectors between E. coli and Bifidobacterium. The plasmid pKJ36 was composed of 3,625 base pairs with a 65.1% G+C content. The structural organization of pKJ36 was highly similar to that of pKJ50, and the three major ORFs on pKJ36 showed high amino acid sequence homologies with those of pKJ50. The putative proteins coded by these three ORFs were designated as RepB (32.0 kDa, pI=9.25), MembB (29.0 kDa, pI=12.25), and MobB (39.0 kDa, pI=IO.66), respectively. The amino acid sequence of RepB showed a 57% identity and 70% similarity with that of the RepA protein of pKJ50. Upstream of the repB gene, the so-called iteron sequence was directly repeated four-and-ahalf times and a conserved dnaA box was identified. An amino acid sequence comparison between the MobB and MobA of pKJ50 revealed a 48% identity and 61 % similarity. A conserved oriT sequence with an inverted repeat identical to that of pKJ50 was also found upstream of the mobB gene. A hydropathy analysis of MembB revealed four possible transmembrane regions. The expressions of the repB and membB genes were confirmed by RT-PCR. The in vitro translation reaction of pKJ36 showed protein bands with anticipated sizes with respect to each putative gene product. S 1 endonuclease treatment and Southern hybridization suggested that pKJ36 replicates by a rolling circle mechanism via a single-stranded DNA (ssDNA) intermediate. A shuttle vector between E. coli and Bifidobacterium sp. was constructed using the pKJ36, pBR322, and staphylococcal chloramphenicol acetyl transferase (CAT) gene. The successful transformation of the Bifidobacterium strains was shown by Southern hybridization and PCR. The transformation efficiency differed from strain to strain and, depending on the electroporation conditions, with a range between $1.2{\times}10^1-2.6{\times}10^2{\;}cfu/\mu\textrm{g}$ DNA.X> DNA.

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Construction and Characterization of a Recombinant Bioluminescence Streptomycetes for Potential Environmental Monitoring

  • Park, Hyun-Joo;Hwang, Keum-Ok;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
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    • v.12 no.4
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    • pp.706-709
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    • 2002
  • Bacterial bioluminescence has been known to be a highly valuable reporter system for its potential application as an effective and simple environmental monitoring method for toxic compounds. In this short report, we constructed a streptomycetes-Escherichia coli shuttle vector-containing bioluminescence system and evaluated its potential application for toxic compounds monitoring. The luxAB biolurninescence genes from Vibrio harveyi were cloned into a streptornycetes-E. coli shuttle vector (named pESK004) and functionally expressed in Streptomyces lividans. The recombinant S. lividans containing pESK004 exhibited an optimal biolurninescence at the optical density ($OD_{600\;nm}$) of 0.4-0.5 and aldehyde concentration of 0.005%. When the recombinant bioluminescence streptomycetes was exposed to a toxic compound such as heavy metals, chlorinated phenols, or pesticides, the bioluminescence was decreased proportionally to the concentration of toxic compound in the assay mixture. The $EC_{50}$ (effective concentration to decrease 50% of the bioluminescence prior to exposure) values in the recombinant biolurninescence streptomycetes for mercury, 2,4-dichlorophenol, and malathion were measured at 2.2 ppm, 144.0 ppm, and 82.4 ppm, respectively. The degree of sensitivity and specificity pattern toward these toxic compounds characterized in this recombinant bioluminescence streptomycetes were unique when compared with previously reported bacterial bioluminescence systems, and this revealed that a recombinant bioluminescence streptomycetes might provide an alternative or complementary system for potential environmental monitoring.