• Mycobiology
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• 제38권1호
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• pp.70-73
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• 2010

Temperature-sensitive yeast mutants were used to screen for cell cycle-related genes from Pleurotus eryngii genomic DNA. A mushroom genomic DNA library was established and each gene was screened for the ability to rescue seven Saccharomyces cerevisiae temperature-sensitive strains. Hundreds of yeast transformants were selected at restrictive temperatures over $30^{\circ}C$. Plasmids from the transformants that survived were isolated and transformed back into their host strains. The temperature sensitivity of the resulting transformants was tested from $30^{\circ}C$ to $37^{\circ}C$. Ten DNA fragments from P. eryngii were able to rescue yeast temperature-sensitive strains, and their DNA sequences were determined.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.427-429
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• 1996

We have already cloned an extracellular $\alpha$-glucosidase gene from Aspergillus niger with oligonucleotide probe synthesized on the basis of the peptide sequences determined previously. The DNA sequence revealed an open reading frame of 895 amino acids split by three introns. We are attempting to construct an A. niger strain deficient in the $\alpha$-glucosidase enzyme activity, which would be useful for the glucoamylase production without contamination by the industrially undesirable $\alpha$-glucosidase. For destruction of the $\alpha$-glucosidase gene, we try to make transformations. A cloned partial $\alpha$-glucosidase gene was introduced into Aspergillus niger, and transformants with suppressed $\alpha$-glucosidase activity were isolated. The transformants were cultured on YPD medium which contained Hygromycin B at 30$\circ$C. The activity of $\alpha$-glucosidase of the suppressed transformants was compared to that of wild type activity. As shown by southern-hybridization, we detected that the transformant was a heterocaryon.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.39-44
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• 2010

Acidithiobacillus caldus is an acidophilic, chemolithotrophic bacterium that plays an important role in bioleaching. Gene transformation into A. caldus is difficult, and only the conjugation method was reported successful, which was a relatively sophisticated method. In this research, electrotransformation of A. caldus species was achieved for the first time using A. caldus Y-3 and plasmid pJRD215. Transformants were confirmed by colony PCR specific to the str gene on pJRD215, and the recovery of the plasmid from the presumptive transformants. Optimizations were made and the transformation efficiency was increased from 0.8 to $3.6{\times}10^4$ transformants/${\mu}g$ plasmid DNA. The developed electrotransformation method was convenient in introducing foreign genes into A. caldus.

• KSBB Journal
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• 제11권4호
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• pp.504-509
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• 1996

Promoters which are useful for constructing expression vectors for lactic acid bacteria were obtained from the chromosomal DNA of Lactococcus lactis ssp. lactis MG1363. pBV5030, a promoter-selection vector, replicates in L. lactis and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene (cat-86). After examining E. coli transformants which grew on LB media containing chloramphenicol (Cm, 20$\mu\textrm{g}$/mL) , many MG1363 derived DNA fragments which encompass promoter sequences were identified. Some recombinant E. coli cells can grow at the Cm concentration of 1,000$\mu\textrm{g}$/mL. When plasmids from those highly resistant E. coli cells were purified and introduced into L. lactis ssp. lactis MG1614 cells by electroporation, lactococcal transformants showing Cm resistance were obtained. So far, five plasmids with different promoter inserts were introduced into L. lactis MGl614 cells. The maximum level of Cm resistance in L. lactis MG1614 transformants was quite low (20$\mu\textrm{g}$/mL) when compared with that observed in recombinant E. coli cells harboring the same plasmids.

• Archives of Pharmacal Research
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• 제21권4호
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• pp.470-474
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• 1998

A kanamycin producer, Streptomyces kanamyceticus IFO 13414 is highly resistant to kanamycin. Cloning of the kanamycin resistance genes in S. lividans 1326 with pIJ702 gave several kanamycin resistant transformants. Two transformants, S. lividans SNUS 90041 and S. lividan. SNUS 91051 showed similar resistance patterns to various aminoglycoside antibiotics. Gene mapping experiments revealed that plasmids pSJ5030 and pSJ2131 isolated from the transformants have common resistant gene fragments. Subcloning of pSJ5030 gave a 1.8 Kb gene fragment which showed resistance to kanamycin. Cell free extracts of S. lividans SNUS 90041, S. lividans SNUS 91051 and subclone a S. lividans SNUS 91064 showed kanamycin acetyltransferase activity. The detailed gene map is included.

• ALGAE
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• 제28권4호
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• pp.379-387
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• 2013

The purpose of this study was to express bovine lactoferrin N-lobe in Chlorella vulgaris, a green microalga, using the pCAMBIA1304 vector. Chlorella-codon-optimized bovine lactoferrin N-lobe (Lfb-N gene) was cloned in the expression vector pCAMBIA1304, creating the plasmid pCAMLfb-N. pCAMLfb-N was then introduced into C. vulgaris by electro-transformation. Transformants were separated from BG-11 plates containing 20 ${\mu}g\;mL^{-1}$ hygromycin. Polymerase chain reaction was used to screen transformants harboring Lfb-N gene. Finally, total soluble protein was extracted from the transformants, and the expression of Lfb-N protein was detected using western blotting. Using this method, we successfully expressed bovine lactoferrin in C. vulgaris. Therefore, our results suggested that recombinant lactoferrin N-lobe, which has many uses in the biomedical and pharmaceutical industries, can be produced economically.

• 한국미생물·생명공학회지
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• 제14권2호
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• pp.125-131
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• 1986

각 효모 숙주 및 vector에 따라 lithium염 처리에 의한 생효모형질전환 최적 조건을 얻기 위하여 5종의 효모 숙주(S. cerevisiae Dl3-lA, DKD-5D, DBY-746, MC-16 및 S2022D)에 3종의 효모plasmid vector(YRp 7, YEp 13 및 YIp 5 )의 형질전환 실험과 아울러 이들 각 형질전환체내에서 도입된 plasmid들의 안정성을 조사하였다. lithium 염의 경우 LiCl가 Li-acetate에 비하여 좋은 효과를 보였으며 LiCl처리에 의한 최적 형질전환 조건은 각 숙주-vector계에 있어서 공히 균체 배양 시간은 16시간 (5.4 $\times$ $10^6$ - 2.4$\times$$10^8$cells/$m{\ell}$ ) 내외, LiCl의 농도는 0.1-0.2M, PEG(4000)의 농도는 35%, induction시간은 60분 내외 열처리는 42$^{\circ}C$에서 5분간, LiCi처리 buffer는 0.1M tris-HCl(pH 7.6)에서 가장 높은 형질전환 빈도를 보였다. 한편 Protoplast 형질전환법과 형질전환빈도를 비교해 본 결과 DKD-5 D(YEp13)과 Dl3-lA(YRp7)의 경우는 protoplast법이 DKD-5D(YRp 7), 및 DBY-746(YEp 13)에서는 LiCl처리법이 형질전환 빈도가 높았으며 MC-16(YEp 13)의 경우는 양방법에서 공히 비교적 낮은 빈도를 보였다. 각 형질전환체내에서의 도입된 plasmid의 안정성은 선택배지에서 배양했을 경우- YRp 7이 Dl3-lA 및 DKD- 5D 숙주에서 70세대후에는 80-85%가 유실되었으나 YEp13은 DKD-5D 및 DBY-746에서 35%밖에 유실되지 않았으며 MC-16숙주에서는 55% 유실로서 비교적 안정하였다. 또한 비선택배지에 배양시에는 선택배지에 배양했을 경우 보다 안정성이 다소 낮았으나 같은 경향은 보였다.

• 한국자원식물학회지
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• 제21권4호
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• pp.260-264
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• 2008

고추 형질전환체 $T_1$ 및 $T_2$세대의 유묘에 역병균을 접종한 결과 유주포자 $10^3$개/mL에서 접종 5일후 역병 발병율은 $T_1-1$계통 4.0%, $T_1-2$ 10.0%이었으며, 접종 12일후는 $T_1-1$ 계통 52.0%, $T_1-2$ 계통 64.0%로 대비종인 '금탑'과 '수비초' 100%에 비해 발병율이 떨어져 유주포자 $10^3$개/mL에서는 형질전환체의 저항성 계통선발이 가능하였다. 획득된 역병 저항성 형질전환체를 영양고추시험장의 역병 상습포장에서 재배한 결과 생육, 개화소요일수, 과특성 등은 계통간에 다소 차이가 있었으나, 과실모양은 수비초와 매우 유사하였다. 포장에서의 역병의 발생은 계통간에 차이가 많았으며, $T_2-1-6$ 계통과 $T_2-4-9$ 계통은 시판종인 금탑에 비해 약 30%정도 적었다. 포장에서 선발된 형질전환체는 세대의 진전을 통한 저항성의 고정이 필요한 것으로 판단된다. 이상의 결과로 본 연구에서 확립된 형질전환 시스템으로 고추의 역병저항성 형질전환체의 육성이 가능할 것으로 생각된다.

• 미생물학회지
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• 제46권1호
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• pp.93-95
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• 2010

리그닌 분해효소 군을 가진 백색부후균들은 다핵방향족 화합물을 포함하여 염료와 폭약 및 내분비장애 물질의 분해 등 다양한 난분해성 물질을 분해할 수 있다. 리그닌 분해효소 중 laccase와 manganese peroxidase (MnP)를 각각 발현하는 벡터를 국내에서 분리한 백색부후균류의 하나인 아교버섯(Phlebia tremellosa)에 형질전환 방법으로 도입시킨 형질전환체를 사용하여 염료의 탈색능력을 분석하였다. Methyl green의 경우 3일 후 약 50%의 탈색을 보인 야생형에 비하여 laccase 형질전환체(TF2-1)와 MnP 형질전환체(T5) 모두 90% 이상의 탈색을 보였다. Remazol brilliant blue R (RBBR)에서는 야생형이 약 67%의 탈색을 보인 반면 두 가지 형질전환체들은 약 85%의 탈색을 보였다. 각각의 형질전환체들은 laccase와 MnP의 활성 및 유전자 발현도 활발한 것으로 확인되었다.

• 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.