• 한국미생물·생명공학회지
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• 제39권4호
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• pp.337-344
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• 2011

이전 연구에서, bacteriophage ${\Phi}FC1$이 Enterococcus faecalis KBL703에서 UV induction을 통해 분리 동정되었으며, ${\Phi}FC1$은 phage attachment site인 attP와 bacterial attachment site인 attB 사이에서 site-specific integration을 촉매하는 integrase를 가지고 있다는 것을 밝혀냈으며 이를 MJ1이라 명명하였다. 이 연구에서는 이를 바탕으로 MJ1에 의한 site-specific integration의 효율을 Escherichia coli와 NIH3T3 cell에서 확인 하기 위해 attP, attB, MJ1을 각각의 벡터에 삽입하였다. MJ1 인테그라제에 의한 재조합을 수행하기 위해서 기질 벡터 pABLP를 $DH5{\alpha}$에 형질전환시킨 후, LB 배지에서 $37^{\circ}C$ 1시간 배양한 후 암피실린(ampicillin)과 테트라싸이클린(tetracycline) 항생제 플레이트로 pGMJ1과 pABLP 같이 가지고 있는 colony 들을 선별하여, LacZ 유전자가 불활성화 된 흰색 콜로니 개수를 세고 통계를 낸 결과 integration의 frequency가 99% 이상인 것으로 나타났다. 또한, 실제로 재조합이 일어났는 지를 확인하기 위해서 콜로니 PCR을 수행하여 재조합의 산물인 attL 150 bp을 확인하였다. PCR 산물은 염기서열분석을 통해 정확한 site-specific integration이 일어났음을 확인하였다. MJ1에 의한 integration을 보이기 위해 attP와 attB를 가지고 있는 vector를 MJ1 expression vector와 함께 NIH3T3 cell에 cotransfection 했으며 GFP를 reporter로 사용해 그 activity를 관찰하였다. NIH3T3 cell에서 GFP의 발현을 형광 현미경을 통해 알아본 결과, MJ1에 의한 sitespecific integration이 다른 accessory protein의 도움 없이 일어난다는 것을 볼 수 있었다. 마찬가지 방법으로, attR과 attL 간의 excision을 GFP로 알아본 결과, GFP는 발현하지 않았으며, 이는 MJ1에 의한 excision이 일어나지 않았음을 보여주었다. 이와 같은 결과로 볼 때, MJ1의 host만이 아니라 넓은 범위안에서도 integration을 수행할 수 있다는 것을 보여주었다. 따라서 MJ1을 이용한 site-specific integration system의 개발은 gene therapy를 위한 gene delivery system의 구축에 있어서 좋은 시작이 될 수 있다.

• BMB Reports
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• 제29권5호
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• pp.448-454
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• 1996

Bacteriophage ${\phi}FC1$ is a temperate phage which was identified as a prophage in the Enterococcus faecalis KBL703 chromosome. Phage ${\phi}FC1$ integrates into the host chromosome by site-specific recombination. The phage attachment site P (attP) was localized within the 0.65-kb XhoI-HindIII fragment and the nucleotide sequence of the region was determined. An open reading frame (mj1) which adjoined the phage attachment site encoded a deduced protein related to the site-specific recombinase family. The organization of this region was comparable to other site-specific recombination systems. The molecular weight of the expressed MJ1 in E. coli was in good agreement with the predicted 53,537 Da of the mj1 gene product. Elucidation of the phage-specific integration process in this study would provide useful genetic tools such as a chromosomal integration system.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.217-228
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• 2009

Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.250-256
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• 2004

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.826-830
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• 2018

A Cre/loxP-${\delta}$-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae. To allow repeated integrations, the reusable Candida glabrata MARKER (CgMARKER) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and ${\beta}$-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

• 한국지진공학회논문집
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• 제20권4호
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• pp.245-256
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• 2016

In the companion paper (I - Database and Site Response Analyses), site-specific response analyses were performed at more than 300 domestic sites. In this study, a new site classification system and design response spectra are proposed using results of the site-specific response analyses. Depth to bedrock (H) and average shear wave velocity of soil above the bedrock ($V_{S,Soil}$) were adopted as parameters to classify the sites into sub-categories because these two factors mostly affect site amplification, especially for shallow bedrock region. The 20 m of depth to bedrock was selected as the initial parameter for site classification based on the trend of site coefficients obtained from the site-specific response analyses. The sites having less than 20 m of depth to bedrock (H1 sites) are sub-divided into two site classes using 260 m/s of $V_{S,Soil}$ while the sites having greater than 20 m of depth to bedrock (H2 sites) are sub-divided into two site classes at $V_{S,Soil}$ equal to 180 m/s. The integration interval of 0.4 ~ 1.5 sec period range was adopted to calculate the long-period site coefficients ($F_v$) for reflecting the amplification characteristics of Korean geological condition. In addition, the frequency distribution of depth to bedrock reported for Korean sites was also considered in calculating the site coefficients for H2 sites to incorporate sites having greater than 30 m of depth to bedrock. The relationships between the site coefficients and rock shaking intensity were proposed and then subsequently compared with the site coefficients of similar site classes suggested in other codes.

• Food Science and Biotechnology
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• 제17권6호
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.

• Geomechanics and Engineering
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• 제34권1호
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• pp.29-41
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• 2023

The 3D geospatial modeling of geotechnical information can aid in understanding the geotechnical characteristic values of the continuous subsurface at construction sites. In this study, a geostatistical optimization model for the three-dimensional (3D) mapping of subsurface stratification and the SPT-N value based on a trial-and-error rule was developed and applied to a dam emergency spillway site in South Korea. Geospatial database development for a geotechnical investigation, reconstitution of the target grid volume, and detection of outliers in the borehole dataset were implemented prior to the 3D modeling. For the site-specific subsurface stratification of the engineering geo-layer, we developed an integration method for the borehole and geophysical survey datasets based on the geostatistical optimization procedure of ordinary kriging and sequential Gaussian simulation (SGS) by comparing their cross-validation-based prediction residuals. We also developed an optimization technique based on SGS for estimating the 3D geometry of the SPT-N value. This method involves quantitatively testing the reliability of SGS and selecting the realizations with a high estimation accuracy. Boring tests were performed for validation, and the proposed method yielded more accurate prediction results and reproduced the spatial distribution of geotechnical information more effectively than the conventional geostatistical approach.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.