• 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.

• KSBB Journal
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• 제19권5호
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• 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.

• 한국미생물·생명공학회지
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• 제36권2호
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• pp.128-134
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• 2008

Erro-prone PCR에 의해서 열안정성이 향상된 Streptomyces coelicolor A(3) 유래의 L-threonine aldolase를 Corynebacterium glutamicum R에서 과잉발현시키기 위하여 Corynebacterium용 vector plasmid인 pCRB1의 SD배열과 개시코든사이의 1염기를 제거한 고발현용 vector plasmid인 pCG-H44(2)를 구축하였다. pCG-H (2)에 의해서 형질전환된 C. glutamicum R 균주(CGH44-2)에서 L-TA를 발현시킨 결과, 기존의 Corynebacterium용 vector plasmid인 pCRB1(CGH44-1)보다 L-TA의 발현량이 높았다. L-threo-DOPS의 합성을 위한 최적조건은 $30^{\circ}C$, 0.1 M cirtric acid buffer(pH 7.0)이었으며, 0.1% TritonX-100를 첨가하였을 경우 보다 높은 활성을 보였다. 최적조건하에서 CGH44-2를 whole cell biocatalyst로 이용한 반복회분식반응에서 재조합대장균을 숙주로 이용한 경우보다 재조합Corynebacterium을 이용하였을 경우, 목적하는 L-threo-DOPS의 합성이 안정적으로 이루어졌다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제6권4호
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• pp.581-589
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• 1993

A plasmid vector, $RSVLTR/{\beta}G2$, containing lacZ gene under the control of the RSVLTR promoter were transfected into chicken embryo fibroblasts by three different transfection methods. Calcium phosphate, lipsome and DEAE-dextran techniques were applied for transfection of chicken cells. A histochemical assay with X-gal was used as a simple method for screening transfected cells. Plasmid $RSVLTR/{\beta}G2$ was expressed proficiently in the chicken embryo fibroblast. Calcium phosphate-DNA precipitate transfection resulted in the highest efficiency for transient expression of $RSVLTR/{\beta}G2$. Transfected cells formed colonies on the 9th day of incubation indicating stable transformation of the inserted plasmid.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.837-842
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• 2013

A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.

• 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.

• 미생물학회지
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• 제33권4호
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• pp.274-276
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• 1997

Coprinus congregatus에서 laccase를 과량생성하는 변이주를 대상으로 phosphinothricin 저항성을 선택표지로 사용하여 형질전환을 수행하였다. 항생물질에 대한 저항성을 부여하는 plasmid DNA(pBARGEM 7-1)를 제한효소로 가수분해하여 원형질체에 형질전환을 수행한 결과 plasmid DNA $1{\mu}g$ 당 약 500여개의 형질전환체를 얻었다. 도입된 plasmid는 형질전환체의 chromosomal DNA에 삽입되어 있음을 Southern blot으로 확인하였다.