• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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• pp.51-51
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• 2018

The application of recombinant DNA technology has been remarkable and nearly replaced commonly used traditional methods. Traditional industrial microbiology long depended on the discovery of valuable strains and mutagenesis of such strains to improve its secretion capacity of enzymes and secondary metabolites on the industrial scale. Commodities included industrial enzymes and biopharmaceuticals. The purpose of genome manipulation by the crossing of different strains or genetic recombination of naked DNA to the genome is of increased production of valuable metabolites. We optimized a transformation method to either for removal of innate genes, introduction of heterologous genes, or combination of both. We have been used selected whole or partial genes to manipulate target fungi toward the development of strains overproducing invaluable proteins. We have also used the whole genome sequence information of fungal genomes in public databases and functional genomics approach to select genes to manipulate and eventually contributing greatly to the development of overproducing industrial strains overproducing proteins or secondary metabolites. I will briefly review 1) filamentous fungi as a host for production of recombinant proteins and secondary metabolites, 2) markets of industrial metabolites, 3) a new approach to manipulate up to five genes at the same time in the system that ProxEnrem uses.

• Journal of Microbiology and Biotechnology
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• 제31권7호
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• pp.903-911
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• 2021

Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed, and future research and development directions are also proposed.

• 한국발생생물학회지:발생과생식
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• 제6권2호
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• pp.71-76
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• 2002

최첨단 연구로 성장하고 있는 해양생물공학 연구 중 염색체공학 연구의 배수체 조작, 성분화, 자성 발생, 형질전환 어류와 유전자 조작에 관한 최근의 연구 동향을 분석하고 향후 발전 방향에 관하여 살펴보았다. 배수체 연구 중 3배체 어류 생산 연구는 20여종의 어류들 중 무지개 송어와 메기가 물리적 충격을 이용한 실험법으로 산업적으로 이용 가능한 수준에 있다. 성분화를 포함한 약 15종의 고급 어종이 방사선 투사법에 의해 자성 발생 2배체를 생산 유도하였다. 형질전환 어류는 1985년 이래 약 40여종의 어류가 외래 유전자를 미세 조작이나 전기충격법에 의해 형질전환되고 있다. 최근에 역바이러스성 벡터들에 의한 외래 유전자를 이용한 형질전환 어류가 생산되었으며 이러한 형질전환 어류는 기초과학 육성은 물론 해양생물공학 연구의 발전이 기대되고 있다.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• 한국가축번식학회지
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• 제20권2호
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• pp.89-99
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• 1996

The purpose of this study was to develop the diagnosis techniques for sex determination of rabbit embryos at preimplantation stage. To detect male specific sequences using polymerase chain reaction, two genes functional on sex determination including SRY and ZFX/Y genes were targeted using multiple oligonucleotide primer sets. Three of them for conserved SRY gene were used for appropriate amplification pattern, and then only one primer set #3 proved to be most efficient, showing male-specific strong signal ofamplified sequences. Using this male specific bandsfrom human, cattle, pig and mouse, the gender of rabbit was determined. As an another system for sex determination system, amplified 910bp fragment from ZFX/Y was digested with several restriction endonuclease and showed gender specific restriction fragments only by Hinf I. Using two different system for sex identification of rabbit in this study, blind tests for 17 samples was conducted and showed identical results from two different methods. And then, amplification limit of PCR reaction for template DNA was estimated using various amounts of DNA for both SRY and ZFX/Y systems, resulted as 20pg and 800pg, respectively. With this results, test for gender identification of rabbit embryos were performed using SRY derived amplification system. From total 22 embryos selected for its developmental state 18 were identified as male embryos, showing significant difference from expected sex ratio 1:1. This biased sex ratio was interpreted as to have been caused by the fact, reported by the fact, reported by several researchers, that male embryos develop more rapidly and are more resistant against the in vitro manipulation than female embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제17권1호
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• pp.68-72
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• 2004

The effects of the addition of yeast on in vitro roughage degradability and methane production were investigated in order to clarify the effects of yeast on the rumen microbes and to establish methods of rumen manipulation. Three roughages (whole crop corn, rice straw and Italian ryegrass) were incubated for 3, 6, 12 and 24 h with or without dried beer yeast following the method described by Tilley and Terry. Using the same method, these roughages were incubated with or without yeast extract, albumin or purified DNA. In vitro methane production was measured with or without dried beer yeast at 12 and 24 h. The degradability of yeast was found to be 57 and 80% at 12 and 24 h, respectively. The rate of degradation of fraction b was 6.16%/h. There was a significant increase in roughage degradability at 6 h (p<0.05), 12 h (p<0.05) and 24 h (p<0.01) by dried yeast addition. The degradability of all three roughages was higher in the samples treated with yeast extract than in the no addition samples except in the case of rice straw incubated for 12 h. Nevertheless, the magnitude of increment was smaller with the addition of yeast extract than without the addition of yeast. With the addition of purified DNA, there were significant increases in roughage degradability at 6 h (p<0.01), 12 h (p<0.01) and 24 h (p<0.05); however, higher degradability values were detected in the samples to which albumin was added, particularly at 6 h. If the degradability values of the no addition samples with those of samples containing yeast, yeast extract, DNA and albumin were compared, the largest difference was found in the samples to which yeast was added, although it is worth noting that higher values were observed in the yeast extract samples than in the DNA or albumin samples, with the exception of the case of rice straw incubated for 24 h. Methane production was significantly increased at both 12 and 24 h incubation. The increment of roughage degradation and methane production brought about by the addition of dried beer yeast to the samples was thought to be due to the activation of rumen microbes. Water soluble fraction of yeast also seemed to play a role in ruminal microbe activation. The increment of degradability is thought to be partially due to the addition of crude protein or nucleic acid but it is expected that other factors play a greater role. And those factors may responsible for the different effects of individual yeast on ruminal microbes.

• 미생물학회지
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• 제34권4호
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• pp.236-242
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• 1998

박테리오파지 P2-P4 시스템은 바이러스 조립과정 기작 연구를 위한 뛰어난 실험 소재로 연구되어 왔으나, 이 시스템에 유전자 조작상 필수적인 유용한 플라스미드가 없는 관계로 그의 필요성이 대두되고 있다. 본연구에서는 도움파지가 없을 때 플라스미드 상태로 존재하는 P4 ash8 (sid71)을 시작물질로, 항생제 내성 유전자를 도입하여 선택성을 줄 목적으로 P4 파지 증식에 불필요한 P4 DNA 단편을 pUC4-K 플라스미드의 kmr(kanamycin resistance) 유전자로 치환하여 P4 ash8(sid71) kmr을 생성하였다. 이 플라스미드는 박테리오파지 P2로 induction하여 박테리오파지 P4 상태로 증식시킬 수 있게 그 genome 크기를 조정하였으며, 파지 상태로 전환된 것을 burst size 결정 및 CsCl 부양 균등밀도 편차실험을 통해 입증하였다. 생성된 P4 플라스미드 유도체를 유전자 조작하여 P4의 integrase 기능을 잃은 변이체를 쉽게 만들 수 있었으며, 역시 박테리오파지 P4 상태로 증식시킬 수 있었다. 이러한 변이체 플라스미드의 안정성 실험을 수행하여, P4의 integrase 기능이 지속적인 플라스미드 상태 유지를 위해 필요하다는 것을 보여주었다.

• Journal of Photoscience
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• 제12권2호
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• pp.75-82
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• 2005

Rumex acetosa L. is a dioecious flowering plant with well developed sex chromosome system: 2n = 12 + XX in the female plants and 2n = 12 + XY1Y2 in the male plants. To isolate sex-linked DNA, we carried out chromosome micromanipulation, followed by DOP-PCR, AFLP of the PCR products, reverse Southern hybridization and sequence analysis. From 500 AFLP specific clones, 13 X-chromosome and 5 Y-chromosome specific clones were obtained. Except one clone RADAX-239 ($\underline{R}umex\;\underline{a}-\underline{D}OP-PCR-\underline{A}FLP-\underline{Y}-chromosome\;specific$), all clones appear to be R. acetosa plant-specific sequences and non-coding sequences. Southern blot analysis using these clones could not discriminate genomic DNAs either from male or female plants. Results of this study imply that both autosome-origin and degeneration of sex chromosomes are prevalent in plant systems.

• 한국정보통신학회논문지
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• 제17권5호
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• pp.1239-1244
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• 2013

임상 의료 분야에서 질병 진단 및 치료를 위해서는 분자 수준(프로틴, DNA 등)의 크기 뿐만 아니라, 세포 수준에 대한 분석이 필요하다. 많은 경우 실험 샘플이 시간에 따라 변질되기 때문에 정확한 분석을 위해서는 빠른 분석과 실시간 데이터가 필요하다. 본 연구에서는 나노 마이크로 크기의 세포내 단백질이나 DNA의 변화 과정 등을 촬영할 수 있는 3차원 형광 관측 장치를 제작하고 이로부터 얻은 형광 이미지를 실시간 통합 관리 및 분석하기 위한 서버 클라이언트 기반의 형광 이미지 분석 시스템을 구축하였다. 시스템은 형광 관측 장치와 소프트웨어 그리고 형광 이미지를 실시간으로 분석할 수 있는 모바일 프로그램으로 구성된다. 개발된 시스템은 의료인이 시공간의 제약 없이 응급환자의 샘플에서 획득한 형광이미지를 실시간으로 전송받아 분석 및 진단을 내릴 수 있도록 해 주므로 유비쿼터스 헬스 구현에 활용할 수 있다.

• 미생물학회지
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• 제51권1호
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• pp.1-6
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• 2015

"P2 sir-관련 도움파지 비효율성"이라는 용어는 P2 sir 변이체가 그들의 위성 박테리오파지인 P4에 대해 도움파지 역할을 충분히 못하는 것을 가리키는 용어이다. 이 연구의 목적은 이러한 P2 sir-관련 도움파지 비효율성을 극복하는 요인들을 조사하는 것이다. 우선 P2의 cos 영역을 함유하는 P4 sid71 cosP2가 P2 sir3의 도움파지 비효율성을 극복하는지를 조사하였다. 그 결과 P4 sid71 cosP2는 P2 sir3의 도움파지 비효율성을 극복하지 못하였다. P2의 cos 영역 대신에 $P2_{sir}$-sized head에 packaging되는 DNA 크기가 도움파지 비효율성을 극복하는 중요한 요인으로 나타났다. 이 연구에서는, DNA 조작을 통해 packaging 되는 DNA 크기가 P4 ost1과 P4 ost2 사이가 되는 세 종류의 P4 유도체를 조성하였다. 그 중 하나인 P4 sid71 delRI::apr의 CsCl 균등밀도차실험을 거쳐 packaging되는 DNA의 크기를 확인하였다. P4 유도체들의 후손방출량 실험에 따르면 그들은 모두 P2sir3-관련 도움파지 비효율성을 극복하였다. $P2_{sir3}$-sized head에 잘 packaging되는 P4 유도체의 크기는 28-29 kb로 나타났다.