• 미생물학회지
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• 제29권3호
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• pp.149-154
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• 1991

Escherichia coli/Corynebacterium glutamicum shuttle vectors, pECCG1 and pECCG2 were constructed by joining a 3.00 kb cryptic plasmid pCB 1 from C. glutamicum and a 3.94 kb plasmid pACYC 177 from E. coli. By trimming unessential parts and introducing mulitiple cloning site into the plasmid pECCG 1, a plasmid pECCG122(5.1kb) was constructed. All the shuttle vectors were stably maintained in C. glutamicum up to about 40 generations irrespective of kanamycin addition in the medium. Threonine operon (homoserine dehydrogenase/homoserine kinase) and dapA gene (dihydrodipicolinate synthetase) of C. glutamicum were cloned into the plasmid pECCG122, and the resultant plasmids were designated pTN31 and pDHDP19, respectively. They were used to study the effect of cloned foreign gene on the stability of the plasmid pECCG122. Plasmids pTN31 and pDHDP19 were segregated rapidly from C. glutamicum when cultured in the medium without kanamycin. In medium with $50\mu${\g/ml} of kanamycin, their segregation rates were much slower than those in medium without kanamycin, but the danamycin addition didn't guarantee the complete maintenance of the plasmids in C. glutamicum.

• 미생물학회지
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• 제25권4호
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• pp.262-273
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• 1987

pBR 322와 pBD9을 이용하여 Staphylococcus aureus에서 분리된 chloramphenicol 저항성(Cmr) plasmid인 pSBK 203상의 ori 부위를 cloning하였다. 또한 E. coli 내에서도 발현하는 pSBK 203상의 Cm 저항성 부위 및 cloning 된 ori 부위를 pBR 322에 재조합시켜 E. coli와 그람양성균인 Bacillus subtilis 양쪽 모두에서 복제되고 또 항생물질에 대한 저항성도 각각 발현되는 shuttle vector 구성을 시도하였다.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1702-1708
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• 2015

We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

• 한국미생물·생명공학회지
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• 제15권5호
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• pp.319-327
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• 1987

알코올 생산성이 높은 Zymomonas 균주의 기질 이용성을 넓히기 위한 목적으로 natural replicon을 포함하며 적당한 항생제 저항표지를 갖는 plasmid vector의 제조를 시도하였다. Z. mobilis ATCC10988에서 분리된 몇 개의 plasmid중 3.9kb의 적당한 크기를 갖는 pZM3를 선정하여 수종의 제한효소로 처리하여 절편의 크기에 따라 유전자 지도를 작성하였다. pZM 3의 replicon과 pBR 325의 chloramphenicol 저항유전자를 포함한 재조합 plasmid인 pHZ22를 개발하고 이 plasmid vector가 숙주세포인 Z. mobilis ATCC31821에서 독립적으로 replication됨을 확인하였다. 또 하나의 항생제 저항표지로서 RP4의 tetracycline 저항유전자를 분리하여 pHZ22에 도입함으로써 pHZT224를 제조하였는데 이 plasmid vector도 Zymomonas로 conjugation에 의해 전이되어 안정하게 유지 되었다. 본 연구를 통하여 개발된 plasmid vector는 Z. mobilis와 E. coli에 공히 작용하는 shuttle vector 로서 외부 유전자를 Zymomonas에 도입시킬 수 있는 유용한 유전자 운반체임이 확인되었다.

• 미생물학회지
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• 제36권2호
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• pp.109-111
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• 2000

Lactobacillus casei ssp. casei NCIB 4114 균주로부터 3,5kb의 플라스미드를 분리하여, 이 플라스미드를 함유하는 Escherichia coli-Lactobacillus 셔틀 백터(shuttle vector)들을 만들었다. tu틀 벡터들은 모두 electroporation에 의해 성공적으로 형질전환 되었다. Electroporation의 최적조건은 벡터DNA 1$\mu$g당 $2{\times}10^5$ 형질전환체의 효율이었고, 이들 벡터의 성공적 도입은 이들 벡터의 유산균에서의 음식등급 벡터로의 사용 가능성을 제시하였다.

• 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.

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.31-36
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• 1991

Tn5의 kanamycin 저항성 유전자를 가진 pBEL1 plasmid와 C.glutamicum cryptic plasmid인 pSR1으로 7.5kb의 새로운 plasmid를 만든 후, 이를 pCE1301이라 명명하였다. 이 pCE1301은 PEG1301은 PEG-protoplast법으로 C.glutamicum을 형질전환하였을 때 효율이 약 $3.0\times 10^3$형질전환제/$\mu g$이었으며 SalI과 EcoRI 제한효소 절단부위가 1개씩이었다. 또 Km이 없는 배지에서 25세대까지 안정하게 유지되었으며 B.flavum, E.coli에서 복제되었다. 이 pCE1301을 이용하여 C.glutamicum 의 homoserine dehydrogenase 유전자를 cloning하였다.

• Journal of Microbiology
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• 제35권2호
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• pp.149-151
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• 1997

A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The numan cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced .betha.-galactosidase, dimonstrating its dual host usage.

• 미생물학회지
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• 제22권4호
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• pp.249-255
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• 1984

To develop the host-vector system for industrial Coryneform bacteria that seemed to be the most suitable microorganisms for molecular breeding of genes involved in the production of amion acids, nucleotides, and other products of industrial interest, broad host range E. coli plasmid R 1162 DNA was transformed into Brevibacterium ammoniagenes and the plasmids pKU6 isolated from a transformant was physically characterized. All other plasmids from the transformed cells except pKU6 exsisted as multimeric forms in Brevibacterium ammoniagenes. The plasmid DNA was retransformed into Corynebacterium glutamicum with a high frequency ($1.32{\times}10^{-1}$ per cell) and maintained stably both in Brevibacterium ammoniagenes and Corynebacterium glutamicum after 100 generations of cultures with 25-30 copy number per cell. The size of both plasmid pKU6 and plasmid R1162 were the same and restriction maps by EcoR I, Ava I, Pst I, Pvu II and Hinc II were also similar.

• Journal of Microbiology and Biotechnology
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• 제13권3호
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• pp.457-462
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• 2003

The complete nucleotide sequence of a plasmid, pMG1, isolated from Bifidobacterium longum MG1 has been determined. This plasmid, composed of 3,862 base pairs with 65.1% of G+C content. harbors two major open reading frames (ORF) encoding putative proteins of 29 kDa (ORF I) and 71 kDa (ORF II). ORF I showed relatively high amino acid sequence homology with replication proteins of other plasmids from Gr Im-positive and -negative bacteria. Upstream of ORF I, four sets of tandem repeat sequences resembling the iteron structure of related plasmids were found. S1 endonuclease treatment and Southern blot analysis revealed that pMG1 accumulates single-stranded DNA (ssDNA) intermediate, which indicate i the rolling circle replication (RCR) mechanism of this plasmid. Homology search indicated that ORF II encodes plasmid mobilization protein, and the presence of highly conserved oriT sequence in the upstream of this gene supported this assumption. RT-PCR showed that only ORF I is expressed in vivo. Based on these results, pMG 1 was exploited to construct a shuttle vector, pBES2. It was successfully transformed into Bifidobacterium and maintained stably.