• 한국식물생명공학회:학술대회논문집
• /
• 한국식물생명공학회 2004년도 춘계학술대회
• /
• pp.49-50
• /
• 2004

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제9권4호
• /
• pp.274-277
• /
• 2004

Production of green fluorescent protein (GFP) as a model foreign protein using different culture scales under co-expression of Vitreoscilla hemoglobin (VHb) in the industrial Escherichia coli strain W3110 (a K12 derivative), was examined. It was found that the VHb co-expressing W3110, exhibited an exceptional and sustained production ability during cell cultures using different scales, while the VHb non-expressing strain showed variable production levels. This high and sustained production ability indicates that the VHb co-expressing E. coli W3110, could be successfully employed for practical large-scale production cultures without the need for serious consideration of scale-up problems.

• 한국정보통신학회:학술대회논문집
• /
• 한국정보통신학회 2012년도 추계학술대회
• /
• pp.994-996
• /
• 2012

본 연구실에서는 새로운 베큘로바이러스 벡터 시스템을 구축하였다. 즉 본 벡터 시스템은 polyhedron promoter, vesicular stomatitis virus G (VSVG), polyA, cytomegalovirus (CMV) promoter, enhanced green fluorescent protein (EGFP), protein transduction domain (PTD)을 코딩하는 유전자들로 구성 되어있다. 이렇게 새로이 제작된 베큘로바이러스 벡터 시스템과 대조군의 벡터 시스템과 효율과 발현율을 비교하였다. 그 결과 본 연구실에서 제작된 베큘로바이러스 벡터 시스템이 다른 대조군의 벡터 시스템에 비해 효과적임을 확인할 수 있었다.

• KSBB Journal
• /
• 제22권1호
• /
• pp.48-52
• /
• 2007

본 연구에서는 sonificator를 장착하여 세포막을 파쇄하고 현장에서 형광을 이용하여 조작이 간편하고 단시간에 DNA와 단백질을 동시에 측정할 수 있는 자동화된 형광기를 개발하기 위하여 단백질을 측정하는데 최적의 파쇄조건을 확립하고자 하였다. 용액 중에 녹아 있는 공기 중의 oxygen은 collisional quenching을 일으키는데 sonification이나 가열처리를 시키면 oxygen이 제거되어 quenching 효과가 크게 감소되어 높은 형광값을 나타내었다. 0.7 X 이상의 형광시약 농도에서는 반응시작 후 1분 이내에 측정해야 하며, 0.3 X 이하의 형광시약 농도에서는 반응시작 후 2$\sim$3분 사이에 반응을 시킨 후 측정하는 것이 바람직한 것으로 나타났다. $100{\mu}g/m{\ell}$ 이상의 BSA 농도에서는 형광시약이 포화되었으며, 시료를 sonification시키면 단백질이 변성되어 눈에 보일 정도로 불투명해져서 시료 용액의 불투명도로 인해 형광 값이 감소되는 경향을 나타내었으며, $1{\mu}g/m{\ell}$ 이하의 BSA 시료에서는 sonification을 시키지 않은 시료보다 sonification을 시켰을 때 $0.125{\mu}g/m{\ell}$의 BSA를 훨씬 더 구분이 잘 되어 낮은 단백질 농도에서는 sonification시키는 것이 훨씬 유리한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
• /
• 제17권10호
• /
• pp.1727-1732
• /
• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• Interdisciplinary Bio Central
• /
• 제4권2호
• /
• pp.5.1-5.9
• /
• 2012

Small peptide tags such as c-myc, HA, or FLAG tag have facilitated efficient Western-blotting of proteins of interest especially when specific antibodies for the proteins are not available. However, the conventional Western-blotting requires the multi-steps process taking at least several hours up to two days. With examples of various applications, here we show a convenient and time-saving method for protein detection which employs a fluorescent chemical BDED and its binding peptide RC-tag. And we propose "Estern staining", as a standard term for protein detection method using fluorescent chemicals and their binding small peptide tags. Eastern staining may substitutes for the time-consuming "immuno-staining" in many versatile applications.

• Parasites, Hosts and Diseases
• /
• 제49권4호
• /
• pp.357-364
• /
• 2011

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

• KSBB Journal
• /
• 제14권3호
• /
• pp.377-379
• /
• 1999

CHO/dhfr- 세포에 대해 LipofectAmine$^{TM}$을 이용한 유전자 이입 효율을 증가시키기 위하여 지질과 DNA의 최적 농도를 구하였다. Reporter 유전자로서 GFP 유전자를 이용하였으며, 여러 농도의 지질 DNA로 유전자 이입된 각 세포군에서 나타나는 green fluorescence intensity를 FACS 분석함으로써 유전자 이입 효율을 정량화 할 수 있었다. 그 결과 24-well plate에서 $2.0{\mu}L$LipofectAmine$^{TM}$과 $0.4{\mu}g$ DNA를 조합하여 사용했을 때 최적의 유전자 이입 효율이 나타남을 알 수 있었다. 또한, GFP는 유전자 이입 최적화를 수행하는 데에 여러가지 면에서 유용한 수단이 될 수 있음을 확인할 수 있었다.

• 한국가시화정보학회지
• /
• 제9권2호
• /
• pp.16-20
• /
• 2011

This paper presents a fabrication method of microcapsules encapsulating fluorescent nanoparticles sensitive to an organic liquid, which is potentially applicable to the encapsulation of protein, cell and drug. It uses the supra-molecular self-assembly of a block copolymer at the interface of the stable and controllable droplets of water suspended with fluorescent nanoparticles and the polymer solved organic. The size and uniformity of the microcapsules were examined for the various polymer concentrations by using SEM image analysis. The maximum standard deviation of the produced microcapsules of less than 3.5% was obtained from the microcapsules produced from the same conditions. The inclusion of fluorescent nanoparticles was visualized in the fluorescence microscope and by using TEM image. It is shown that this fabrication method can provide the uniform size microcapsules with a higher inclusion.

• Journal of Plant Biotechnology
• /
• 제7권2호
• /
• pp.97-103
• /
• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.