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젖산균의 Plasmid DNA 분리방법과 형질전환에 관한 연구
김선기,김현욱 한국유가공기술과학회 1997 Journal of Dairy Science and Biotechnology (JMSB) Vol.15 No.2
This study was carried out to investigate the simpler and faster method for plasmid DNA isolation and the electrotansformation of vector plasmid into lactic acid bacteria. The factors affecting the electroporation efficiency of lactic acid bacteria were studied together. The results obtained were as follows ; 1. O'Sullivan & Klaenhammer's method and its modified method were found to be simpler and faster for plasmid DNA isolation from lactobacilli cells. 2. The presence of plasmids was identified by the modified O'Sullivan & Klaenhammer's method in 4 of 9 L. casei strains, 2 of 9 L. acidophilus strains, 2 of 3 L. delbruekii var. bulgaicus strains, 1 L. brevis strain, and 1 of 2 L. plantarum strains, respectively. 3. The plasmids of lactococcal cells were easily isolated without mutano- lysin treatment. Four plasmids in L. lactis ssp. lactic KCTC2184 and 2 plasmids in Ent. faecalis ATTCC11729 were found. 4. The MPS membrane filtering for E. Coli plasmid isolation was applicable for the isolation of pLZ12 plasmid in L. casei 102S, but the membrane permeability was diminished by the cell debris. 5. Two plasmids in L. lactis ssp. lactis PR3, 3 plasmids in L. fermentum CG1, and 2 plasmids in L. plantarum PA2 were found, respectively. 6. plasmids pBS19 and pBR322 in E. coli JM109 could be directly transferred into E. coliDH5α cells by electroporation at 12.5kV/cm. With the electrotransformation efficiencies of 3.0×10³ and 5.0×10³ cfu/5㎖ of donor cell, respectively, while the pure plasmids were introduced into host cell with the electrotransformation efficiencies were 2.6×10^7 and 1.0×10^8 cfu/㎍ of plasmid DNA, respectively. 7. The cell damages of L, lactis spp. lactis IDM, LM0230 and MG 1363were about 90% at 12.5kV, 90% at l0.0kV and 90% at 10.0kV/㎝, respectively. The electrical damages of 4L. acidophilus strains and L. casei 102S were about 80% and 1% at 12.5kV/㎝ respectively. 8. The survivals of L. lactis ssp. lactis LM0230, MG1363 and IDM after lysozyme treatment for 15 and 45 minites were 50 and 30%, 75 and 60% and 80 and 40%, respectively. 88, 75 and 63% of L. acidophilus WIEBY, HY7008 nd NCFM cells survived after lysozyme treatment for 45 minites. 64, 14 and 14% of L. acidophilus HY7001 survived after lysozyme treatment for 15, 30 and 45 minites. The survival of L. casei 102S was 100% after lysozyme treatment for 45 minites. 9. The electrotransformants of L. lactis ssp. lactis LM0230, MG 1363, IDM were 8.0×10⁴, 3.2×10³, and 3.6×10³ cfu/㎍ of pLZ12 when shocked at 6.0kV, and 3.6×10², and 6.0×10¹ cfu/㎍ of pLZ12 when shocked at 12.5kV respectively. 10. The electrotransformants of lysozyme-treated L. lactis ssp. lactis LM 0230, IDM, MG1363 were 1.2×10^5, 3.8×10³, 5.2×10^5 cfu/㎍ of pLZ12, respectively. 11. The electrotransformants of L. acidophilus WIESBY, NCFM, HY7008, HY7001 were 2.0×10², 7.2×10¹, 3.0×10¹, 8.0×10° cfu/㎍ of pLZ12 at 6.0 kV, respectively. The electrotransformants of L. acidophilus WIESBY and NCFM were not found at 12.5KV, while the L. acidophilus HY7008 and HY7001 gave 3.0×10° and 5.0×10° cfu of electrotransformants. 12. The electrotransformants of lysozyme-treated cells of L. acidophilus WIESBY, NCFM, HY7008, HY7001 were 3.7×10³, 5.6×10², 3.210¹and 4.0׳ cfu/㎍ of pLZ12 at 6.0kV, respect-ively, showing lysozyme treatment increases the efficiency. 13. The electrotransformansts of L. lactis ssp. lactis LM0230, MG 1363, IDM with the Gene Pulser and Progenitor Ⅱ were 8.0×10⁴ and 2.0×10¹ cfu, 3.2×10³ and 1.5×10¹cfu, 3.6×10³ and 1.5×10¹cfu/㎍ of pLZ12, respectively, showing the Gene Pulser indicated higher efficiency than Progenitor Ⅱ. The electrotransformants of L. acidophilus HY 7008 and HY7001 with the Gene Pulser and Progenitro Ⅱ were 3.0×10° and 3.0×10¹cfu, 2.0×10° and 8.0×10¹cfu/㎍ of pLZ12, respectively. With L. acidophilus WIESBY and NCFM, the electrotransformants were not found with Progenitor Ⅱ, while the electrotransformants with Gene Pulser were 2.0×10² and 7.2×10¹cfu/㎍ of pLZ12, respectively. 14. The electrotransformation efficiencies of L. casei 102S were 1.0×10³, 2.1×10³, 3.8×10³ and 1.5×10²cfu/㎍ of pLZ12 at 5.0, 7.5, 10.0 and 12.5kV, respectively. 15. The electrotransformation efficiencies of L. casei 102S cells grown to O.D(A_600)) values of 0.5, 0.8, 1.0 and 1.2 were 5.0×10¹, 8.0×10³, 3.8×10³,and 3.0×10³cfu/㎍ of pLZ12, respectively. 16. The electrotransformation efficiencies of L. casei 102S cells suspended in 10% glycerol, EB and dd H₂O were 3.8×10³, 5.0×10² and 1.5×10² and 1.5×10² cfu/㎍ of pLZ12, respectively, but in 1.0mM HEPES or TE buffer, the electrotran- sformation was not found. 17. At the plasmid DNA concentration of 0.5, 1.0, 2.0 and 3.0㎍ of pLZ12, the electro-transformation efficiencies were 2,0×10³, 3.8×10³, 4.2510³ and 5.07×10³ cfu/㎍ of pLZ12, respectively. 18. The highest electrotransformation efficiency of L. casi 102S was 3.8×10³ cfu/㎍ of pLZ12 with the cells at late-exponential growth phase(O.E A_(600)=1.0) in 10% glycerol by shocking at 10kV/㎝, 200Ohm and 25μFD. 19. Not like L. acidophilus strains, the L. casei 102S cells treated with lysozyme did not enhance the electrotransformation efficiency. 20. When the L. casei 102S cells in 10% glycerol and EB were stored under -20℃ for 1 day or 7 days, the electrotransformation efficiencies were 2.0×10¹ and 1.2×10² and 1.0×10¹ cfu/㎍ of pLZ12, respectively. But the fresh cells in same buffer gave 3.8×10³ and 5.0×10² cfu/㎍ of pLZ12.
The Tuning of the KEK-PS K2 Beam Line for the Hybrid-Emulsion Experiment
Ieiri, M.,Kim, J.Y.,Kim, T.I.,Song, J.S.,Park, I.G.,Chung, K.H.,Kim, C.O.,Park, J.N.,Bahk, S.Y.,Kim, S.K. 慶尙大學校 기초과학연구소 1988 基礎科學硏究所報 Vol.4 No.-
We have shortened the downstream part of the K2 beam line from the original design to raise the kaon yields for the requirement. A layout of the modified K2 beam line is shown in fig. I and the total beam length is 25.7m. Since the high beam flux is not required. Q5 which made the beam flux is not required. Q5 which made the beam achromatic at FF, is omitted. The drift length between Q5 and Q7 is adjusted, so that the total length becomes short and that the beam ellipse at FF satisfies the above condition.
김선기,원송대,연정웅 제주대학교 농과대학 제주도축산문제연구소 1991 畜産論叢 Vol.6 No.1
40 three way crossbreed(LYXHXD) were used to investigate the effect of L. cicidopkilus, Str. faecalis(domestic products as animal probiotics) on the gain, feed efficiency, frequency of diarrhea, fecal lactobacillus and coliform bacteria for 2 months. The results were as follows; 1. ADG(Average Daily Gain) was significantly improved by the addition of L. acidophilus(p<O.l) compared to controL But, significant difference was not found in Str. faecalis treatment 2. Although significant difference was not tested in feed efficiency, there was similar to ADG. 3. Frequency of diarrhea was highly scored in early weanling pigs(0-2 weeks) of all treatments Thereafter, it was not found the diarrhea 4. Counting the fecal lactobacillus and coliform bacteria, it was not found the increase of fecal lactoba-cillus compared to control. Decrease of coliform bacteria was clear at last stage(5-8 Weeks) com-pared to early stage(0-4 weeks).