• Journal of Life Science
• /
• v.18 no.1
• /
• pp.136-142
• /
• 2008

A recombinant Pichia pastoris carrying double expression cassette of Rhodotorula glutinis epoxide hydrolase(RgEH) gene was developed and used for preparing enantiopure (S)-styrene oxide from racemic mixture of styrene oxide. BglII restriction site of original RgEH gene (pPICZ B/RgEH #2) of previous report was mutated using PCR technique for the construction of double expression cassette containing promoter ($P_{AOX1}$), EH gene and transcription terminator ($TT_{AOX1}$) in pPICZ C vector. Double expression cassette with RgEH was inserted into the chromosomal DNA of P. pastoris. $V_{max}$ ($2.2{\mu}mol\;min^{-1}mg\;dcw^{-1}$) on (R)-styrene oxide of P. pastoris with double expression cassette was about 6-fold higher than that ($0.4{\mu}mol\;min^{-1}mg\;dcw^{-1}$) of P. pastoris with single expression cassette. For the determination of the optimal condition, the effects of detergent and temperature on the enantioselective hydrolytic activity and yield of the enantiomer were investigated. When the reaction was performed at $10^{\circ}C$ for 10 min in the presence of 0.5% Tween 20, enantiopure (S)-styrene oxide with 99.9% ee was obtained as the yield 43.4 % from 20 mM racemic sustrate.

• Proceedings of the Korean Society of Precision Engineering Conference
• /
• 2012.10a
• /
• pp.445-446
• /
• 2012

• Journal of the Korea Society for Simulation
• /
• v.22 no.4
• /
• pp.83-92
• /
• 2013

The demand of a camera-lens-module which is installed in mobile phone has been increased explosively as the increase of mobile phone market. Recently, two missions are given to the parts manufacturer of lens module, and they are how to keep the quality of injection moulding process as the increase of resolution, and how to decrease manufacturing cost. In this paper, a simulation study is introduced which is used for developing barrel and shield considering the double-cassette type of mould. At first, the simulation for injection process using Mold Flow$^{TM}$ is applied in the phase of mould design, and mechanical simulation using DPM Assembly$^{TM}$ is applied for collision detection between picking robot and mould. As a result, the productivity increased more than 300%.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.2
• /
• pp.335-341
• /
• 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
• /
• v.11 no.1
• /
• pp.61-64
• /
• 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.

• Development and Reproduction
• /
• v.13 no.3
• /
• pp.191-198
• /
• 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
• /
• v.14 no.5
• /
• pp.1047-1051
• /
• 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
• /
• v.40 no.5
• /
• pp.690-696
• /
• 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
• /
• 2001.11a
• /
• pp.99-102
• /
• 2001

Recombinant Saccharomyces cerevisiae strains harboring various copy numbers of hirudin gene were developed to study dependency of hirudin expression level on its gene copy number. A linear relationship between the copy number of hirudin expression cassette and hirudin expression level was observed up to 10 copies. A double <5-integration vector truncated wi 디 1 the unnecessary bacterial genes before yeast transformation showed a four-fold increase in transformation efficiency and a 1.3-fold enhancement in hirudin expression level compared with a single <5 system. Gratuitous hirudin expression strain was developed by disrupting the GALl gene of S. cerevisiae. Glucose that was fed in a limited manner effectively supported cell growth and hi겨din expression by the gratuitous strain. Effects of methanol concentrations on hirudin production in recombinant Hansenula polymorpha were investigated in continuous and fed-batch cultures. At a steady-state of continuous culture, an optimum methanol concentration of 1.7 g/L was determined at a dilution rate of 0.18 $h^{-1}$ with 1.8 mg/L ${\cdot}$ h hirudin productivity.

• Horticultural Science & Technology
• /
• v.35 no.3
• /
• pp.323-332
• /
• 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.