• 생명과학회지
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• 제18권1호
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• pp.136-142
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• 2008

Rhodotorula glutinis epoxide hydrolase (EH) 유전자를 pPICZ vector에 이중발현 cassette로 재조합하여 발현시킨 재조합균주 Pichia pastoris를 제작하였으며 라세믹 styrene oxide 혼합물로부터 고순도 광학활성 (S)-styrene oxide를 제조하는데 사용하였다. 본 연구에서 사용된 R. glutinis EH 유전자는 전보에서 사용한 pPICZ B/RgEH plasmid DNA를 주형으로 하여 얻었으며 PCR 방법으로 BglII 제한자리를 돌연변이 시키고 AOX1 promoter $(P_{AOX1})$-RgEH 유전자-전사종결서열($TT_{AOX1}$)을 이중으로 가진 이중발현 cassette를 만들어 P. pastoris의 염색체 DNA에 삽입시켰다. 반응온도를 $30^{\circ}C$로 하였을 때, RgEH를 이중발현 cassette로 발현시킨 재조합균주 P. pastoris의 (R)-styrene oxide에 대한 $V_{max}$ 값은 $2.2{\mu}mol\;min^{-1} (mg\;dcw)^{-1}$으로 단일발현 cassette로 발현시킨 P. pastoris의 $0.4{\mu}mol\;min^{-1}(mg\;dcw)^{-1}$에 비하여 6배 향상되었다. 광학순도가 높은 (S)-styrene oxide를 제조하는 최적조건을 찾기 위하여 입체선택적 가수분해 속도 및 수율에 미치는 detergent 및 온도의 효과를 실험하였으며, Tween 20을 0.5% 첨가하고 $10^{\circ}C$로 반응시킨 경우 10분 반응을 통해 99.9% ee 이상의 고순도 (S)-styrene oxide를 43.4% 얻을 수 있었다.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2012년도 추계학술대회 논문집
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• pp.445-446
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• 2012

• 한국시뮬레이션학회논문지
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• 제22권4호
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• pp.83-92
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• 2013

휴대폰 시장의 확산으로 인하여 휴대폰에 장착되는 카메라 렌즈모듈의 수요도 급증하고 있다. 렌즈모듈 부품의 경우 해상도의 증가에 따른 사출공정의 품질보증과 원가절감이라는 두 가지 목표를 달성해야 한다. 따라서 본 논문에서는 더블카세트 방식의 금형구조를 이용하여 경통과 쉴드 두 종류의 제품을 개발하기 위해 시뮬레이션을 활용한 사례를 소개한다. 개발과정에서 두 종류의 시뮬레이션 기술을 활용하였는데, 첫 번째는 금형 설계단계에서 사출품의 성형성을 분석하기 위한 시뮬레이션(일명 Computer Aided Engineering)을 Mold Flow$^{TM}$를 사용하여 수행하였고, 두 번째는 사출기에 금형을 장착한 후 제품이 사출되어 취출하는 동작을 분석하기 위한 동작 시뮬레이션을 DPM Assembly$^{TM}$를 이용하여 수행하였다. 개발 결과 기존 방식에 비해 제품의 생산성이 300% 이상 증가하는 효과를 얻었다.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.61-64
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• 2001

Delta-integration vectors were constructed for the purpose of achieving homologous integration of the hirudin expression cassette into the chromosome of Saccharomyces cerevisiae. A double $\delta$ system truncated with the unnecessary bacterial genes, and consequently having a reduced insert size for integration, showed a four-fold increase in transformation efficiency at given DNA concentrations, and as a result, the constructed recombinant yeast strain had a 1.3-fold enhancement in hirudin expression level compared with a single $\delta$ system.

• 한국발생생물학회지:발생과생식
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• 제13권3호
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1047-1051
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• 2004

The phytopathogenic bacterium Pectobacterium chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzyme such as pectate lyase and cellulase. The cel gene, existing in tandem with the pel gene, was isolated previously [10]. The role of Cel5Z and PelL1 in P. chrysanthemi PY35 pathogenicity on potato tissues was assessed by mutagenizing cloned cel gene and pel gene in tandem and recombining them with the chromosomal alleles. Strains with the Km cassette interposon in pelL1 or a double mutant showed a delay in the appearance of symptoms, suggesting that P. chrysanthemi PY35 pectate lyase PelL1 may playa minor role in soft-rot pathogenesis.

• BMB Reports
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• 제40권5호
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• pp.690-696
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• 2007

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased $K_m$ values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.99-102
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• 2001

본 연구에서는 히루딘을 생산할 수 있는 재조합 S. cerevisiae 에서 ‘히루딘 유전자의 copy number 와 히루딘 발현양과의 관계를 규명하였으며 , ${\delta}$ 서열을 이중으로 사용한 히루딘 발현벡터를 제조하여 히루딘 유전자의 효모염색체로의 도입효율을 증가시켰다. 숙주세포인 효모의 GALl 유전자를 파쇄하여 균체에 의한 갈락토스 소모를 방지하여 보다 경제적으로 히루딘을 생산할 수 있는 시스템을 개발하였으며, 재조합 H. polymorpha을 이용한 발효공정에서 히루딘 생산을 위한 최적의 메탄올 농도를 결정하였다.

• 원예과학기술지
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• 제35권3호
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.