• Food Science and Preservation
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• v.20 no.2
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• pp.250-256
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• 2013

To verify the multiplication of microorganisms on the surface of strawberries, the fate of E. coli $DH5{\alpha}::gfp$ at different temperatures, times and strawberry extract concentrations were measured. The population of E. coli $DH5{\alpha}::gfp$ rapidly increased by 7.36~7.78 log CFU/g at $25{\sim}30^{\circ}C$ for 24 hr and slowly increased by 6.49~8.49 log CFU/g at $10{\sim}20^{\circ}C$ for 48 hr. However, E. coli $DH5{\alpha}::gfp$ did not grow at $10{\sim}15^{\circ}C$ on the surface of the strawberries, regardless of the contact times with the bacterial suspension. E. coli $DH5{\alpha}::gfp$ reached 1.52~3.26 log CFU/g at $20^{\circ}C$ as the contact frequency increased from two to six times. The contact frequencies did not significantly differ. In the case of the six-time contact on the surface of the strawberry at 25 and $30^{\circ}C$, the E. coli $DH5{\alpha}::gfp$ increased by 5.17 and 5.01 log CFU/g. The effects of the strawberry extracts on the growth of E. coli $DH5{\alpha}::gfp$ showed that sterilization and non-sterilization do not affect the growth of microorganisms for 96 hr. In the minimal broth, the growth of E. coli $DH5{\alpha}::gfp$ increased by 1 log CFU/g for 96 hr. In less than 50 percent of the strawberry extracts, the growth rate of E. coli $DH5{\alpha}::gfp$ was higher than in the control and increased by 4 and 5 log CFU/g at 50 and 25 percent of strawberry extracts, respectively. Therefore, E. coli $DH5{\alpha}::gfp$ can multiply and survive on the surface of strawberries when it comes into contact with the fruit extract.

• Proceedings of the Korean Society of Life Science Conference
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• 2003.05a
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• pp.38-47
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• 2003

처음으로 real time으로 유전자 발현을 visualization할 수 있는 marker를 이용할 수 있게 되었다. 이 maker가 바로 green fluorescent protein(GFP; 녹색형광단백질)이다. GFP는1962년 Shimomura 등에 의하여 해파리인 Aequorea victoria에 존재함이 밝혀졌다(1). 그러나 30년이 지난 1992년이 되어서야 Plasher등에 의하여 GFP의 cDNA가 클로닝 되었고(2) 이후 지금까지 약 10년 동안 GFP는 생명과학분야에서 가장 각광받는 유용한 단백질 중의 하나가 되어 매우 다양한 연구에 응용이 되고 있다. 이렇게 GFP가 생명과학분야에서각광을 받게 된 이유로는 GFP가 모든 외래의 세포에서 형광을 발할 수 있는 활성을 가진 형태로 발현되기 때문이다(3). Chalfie 등은 처음으로 GFP를 대장균과 Caenorhabditis elegans에서 발현시켜 유전자의 발현을 모니터함으로써 GFP를 원핵 및 진핵세포 모두에서 사용할 수 있다는 것을 보고하였다(3), 이러한 발견을 통하여 GFP는 살아있는 세포, 조직 및 생물체에 해를 주지 않고 (non-invasive) 유전자의 발현을 측정하는 marker로서 사용할 수 있게 되어 cell biology, developmental biology, neurobiology 및 cytology 등의 연구분야에 널리 이용되고 있다.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.42-42
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• 2003

복제기술은 기존의 형질전환 동물 생산의 효율을 향상시킬 수 있는 기술로서 인정하고 있으며 또한 이를 이용하여 형질전환 동물의 생산이 이루어지고 있다. 따라서 본 연구는 표지유전자 (GFP)와 유용유전자 (hFSH)를 이용하여 임신 45일령에 채취한 태아섬유아세포에 transfection 하고, transfection 된 세포의 효율적인 선발과 이를 이용한 형질전환 복제 수정란을 생산하고자 실시하였다. 대조구 (KbFF), GFP (79KbFF-GFP c-3) 및 hFSH (79KbFF-hFSH n-1)에 공시한 세포는 모두 동일한 태아유래의 세포 (모 79, 부 KPN178,♂)를 이용하였다. pAB-eGFP와 hFSH 유전자는 각파 electroporation 방법을 이용하여 transfection 하고, 이를 2주 동안 G418로 배양하며 selection 하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.1
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• pp.6-10
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• 1998

A plasmid vector containing two reporter genes, mer-lux and lac-GFP, was transformed to both Escherichia coli and Pseudomonas putida. Their cellular activities and biofilm characteristics were investigated in flow-cell units by measuring bioluminescent lights and fluorescent levels of GFP. Bioluminescence was effective to monitor temporal cell activities, whereas fluorescent level of GFP was useful to indicate the overall cell activities during biofilm development. The light production rates of E. coli and P. putida cultures were dependent upon concentrations of HgCl2. Mercury molecules entrapped in P. putida biofilms were hardly washed out in comparison with those in E. coli biofilms, indicating that P. putida biofilms may have higher affinity to mercury molecules than E. coli biofilms. It was observed that P. putida expressed GFP cDNA in biofilms but not in liquid cultures. This may indicate that the genetic mechanisms of P. putida were favorably altered in biofilm conditions to make a foreign gene expression possible.

• Animal cells and systems
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• v.9 no.3
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• pp.107-111
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• 2005

C. elegans DLK-1 has been reported to play an important role in synaptogenesis by shaping the structure of presynaptic terminal. In this study, we investigated the expression pattern and regulation of the dlk-1 gene in C. elegans. To determine the expression pattern, we made a dlk-1::gfp fusion construct, named pPDdg1, which consisted of -2.2 kb 5' upstream region, the first exon, the first intron, and a part of the second exon of the dlk-1 gene. By microinjecting this construct into the worm, we observed that the DLK-1::GFP was expressed mainly in neurons. We next examined the regulatory elements of gene expression by deletion analysis of pPDdg1. Removal of a large portion of the 5' upstream region (${\Delta}-361$ to -2246) of the gene had little effect on the expression pattern, whereas deletion of the first intron led to elimination of the DLK-1::GFP expression in most of the neurons. Our results suggest that the first intron of the C. elegans dlk-1 gene contains the regulatory element critical for gene expression.

• KSBB Journal
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• v.14 no.3
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• pp.377-379
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• 1999

In order to improve the transfection efficiency of CHO/dhfr- cells using cationic lipid, optimal concentrations of the cationic lipid($LipofectAmine^{TM}$) and DNA(pEGFP-C1) need to be determined. The use of green fluorescent protein(GFP) gene as a reporter gene facilitated the quantification of transfection efficiency. The green fluorescence intensity of each cell transfected at various lipid-DNA concentrations was measured using fluorescence-activated cell sorter(FACS) analysis. A combination of $2.0{\mu}L$ cationic lipid and 0.4{$\mu}g$ DNA in a well resulted in the highest trasfection efficiency. Taken together, the method using FACS analysis of GFP is simple and fast, facilitating the optimization of transfection.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.141-146
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• 2005

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.232-234
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• 2002

Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. This study focuses on the production of PLC by B. cereus and recombinant E. coli with fusion protein gene (plc::gfp). Fermentation processes have been monitored by a 2-dimensional fluorescence sensor.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.105-109
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• 2008

We studied on the function and localization of fission yeast Schizosaccharomyces pombe Nup97p, which is homologous to nucleoporin Nic96p in budding yeast Saccharomyces cerevisiae. There was no effect on growth and $poly(A)^{+}$ RNA distribution of cells when nup97 gene was overexpressed. However, the haploid ${\Delta}nup97::kan^{r}$ null mutants confirmed extensive $poly(A)^{+}$ RNA accumulation in the nucleus, abnormal DNA distribution, and cessation of growth when nup97 expression was repressed. We determined the subcellular localization of Nup97 tagged at the N terminus or the C terminus with GFP. Both fusions complemented growth defect of ${\Delta}nup97::kan^{r}$ null mutants. An integrated version of the nup97-GFP fusion was constructed at the nup97 locus. Nup97-GFP fusions expressed from its own promoter was localized at the nuclear periphery with a punctate appearance. These results suggest that Nup97p in fission yeast is also nucleoporin, which is involved in mRNA export.