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    Studies on the function and optical properties of light-regulated YtvA

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    ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์›
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    ํ•™์œ„๋…ผ๋ฌธ (์„์‚ฌ)-- ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› : ์•ฝํ•™๋Œ€ํ•™ ์•ฝํ•™๊ณผ, 2016. 2. ์ด์ง€์—ฐ.YtvA๋Š” flavin mononucleotide(FMN)์„ ๋ณด์œ ํ•˜๋Š” light-oxygen-voltage(LOV) ๋„๋ฉ”์ธ ๋‹จ๋ฐฑ์งˆ๋กœ์จ Bacillus subtilis์—์„œ ์ฒญ์ƒ‰๊ด‘์— ๋ฐ˜์‘ํ•˜์—ฌ ์ผ๋ฐ˜์ ์ธ ์ŠคํŠธ๋ ˆ์Šค ๋ฐ˜์‘์„ ์กฐ์ ˆํ•œ๋‹ค. YtvA๋Š” ์ˆ˜์šฉ์•ก์—์„œ dimer๋กœ ์กด์žฌํ•˜๋ฉฐ, ๋‹ค๋ฅธ LOV ๋„๋ฉ”์ธ ๋‹จ๋ฐฑ์งˆ์— ๋น„ํ•ด ๋น›์— ์˜ํ•œ ๊ตฌ์กฐ ๋ณ€ํ™”๊ฐ€ ๋งค์šฐ ์ž‘๋‹ค๊ณ  ์•Œ๋ ค์ ธ ์žˆ๋‹ค. ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” YtvA์˜ Jฮฑ-helix์˜ ๊ธฐ๋Šฅ๊ณผ ํ˜•๊ด‘ ๋ฆฌํฌํ„ฐ ๋‹จ๋ฐฑ์งˆ๋กœ ์‚ฌ์šฉํ•˜๊ธฐ ์œ„ํ•œ genetic engineering ์‹คํ—˜์„ ์ˆ˜ํ–‰ํ•˜์˜€๋‹ค. Jฮฑ-helix์˜ ๊ธฐ๋Šฅ ์—ฐ๊ตฌ๋ฅผ ์œ„ํ•˜์—ฌ ํ•ด๋‹น ๋ถ€๋ถ„์„ ์ ˆ๋‹จ์‹œํ‚ค๊ฑฐ๋‚˜ ๋Œ€์ฒดํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์˜ ๋น›์— ์˜ํ•œ ๊ตฌ์กฐ๋ณ€ํ™”๋ฅผ native gel๋กœ ๋ถ„์„ํ•˜์˜€๊ณ , Jฮฑ-helix์˜ ์œ ๋ฌด์— ๋”ฐ๋ผ dark regeneration kinetic๊ณผ FMN๊ณผ์˜ ๊ฒฐํ•ฉ๋ ฅ ๋ณ€ํ™”๋„ ๊ด€์ฐฐํ•˜์˜€๋‹ค. ์‹คํ—˜ ๊ฒฐ๊ณผ Jฮฑ-helix๊ฐ€ ์—†๋Š” YtvA ๋‹จ๋ฐฑ์งˆ์€ dark ์ƒํƒœ์—์„œ๋Š” dimer๋กœ, light ์ƒํƒœ์—์„œ๋Š” monomer๋กœ ์กด์žฌํ•˜๋ฉฐ dark regeneration ์‹œ๊ฐ„์€ ๋นจ๋ผ์ง€๊ณ  FMN๊ณผ์˜ ๊ฒฐํ•ฉ๋ ฅ์ด ์•ฝํ•ด์ง€๋Š” ๊ฒฐ๊ณผ๋ฅผ ๋ณด์˜€๋‹ค. YtvA๋ฅผ ํ˜•๊ด‘ ๋ฆฌํฌํ„ฐ ๋‹จ๋ฐฑ์งˆ๋กœ ์„ธํฌ ์ด๋ฏธ์ง•์— ํ™œ์šฉํ•˜๊ธฐ ์œ„ํ•ด์„œ๋Š” ์žฅํŒŒ์žฅ(650 nm ์ด์ƒ)์˜ ๋น›์„ ๋ฐฉ์ถœํ•˜๋Š” ๊ฒƒ์ด ์ค‘์š”ํ•˜๋‹ค. ๋”ฐ๋ผ์„œ random mutagenesis ๋ฐฉ๋ฒ•์„ ์ด์šฉํ•˜์—ฌ directed evolution์„ ์ง„ํ–‰ํ•˜์˜€๊ณ , ๋ฐ˜๋ณต์ ์ธ mutagenesis ์‹คํ—˜ ๊ฒฐ๊ณผ ์žฅํŒŒ์žฅ ์ชฝ์œผ๋กœ ํ˜•๊ด‘์ด ๋ณ€ํ™”ํ•˜๋Š” ๋‹จ๋ฐฑ์งˆ์˜ ํŠน์ • ์œ„์น˜๋ฅผ ๋ฐœ๊ฒฌํ•˜์˜€๋‹ค. YtvA๋Š” GFP ๊ณ„์—ด ๋‹จ๋ฐฑ์งˆ์— ๋น„ํ•ด ์‚ฐ์†Œ์— ๋น„์˜์กด์ ์ด๊ณ  ํฌ๊ธฐ๊ฐ€ ์ž‘๊ธฐ ๋•Œ๋ฌธ์— ์žฅํŒŒ์žฅ์˜ ๋น›์„ ๋ฐฉ์ถœํ•œ๋‹ค๋ฉด ์„ธํฌ๋‚ด์˜ ์‹œ๊ณต๊ฐ„์  ๋ณ€ํ™”๋ฅผ ์ •ํ™•ํ•˜๊ฒŒ ๊ด€์ฐฐํ•  ์ˆ˜ ์žˆ๊ณ , ์‘์šฉ ๋ฒ”์œ„ ๋˜ํ•œ ๋„“์–ด ์งˆ ์ˆ˜ ์žˆ๋‹ค. ๋”ฐ๋ผ์„œ ๋ณธ ์—ฐ๊ตฌ๊ฒฐ๊ณผ๋Š” ๋น›์— ์˜ํ•ด ์œ ๋„๋˜๋Š” ์„ธํฌ ๋‚ด ์กฐ์ ˆ ์‹ ํ˜ธ ๋ณ€ํ™”์— ๋Œ€ํ•œ ์ดํ•ด์™€ ๋‹ค์–‘ํ•œ biotechnology ๊ฐœ๋ฐœ์— ์ดˆ์„์ด ๋  ๊ฒƒ์œผ๋กœ ๊ธฐ๋Œ€๋œ๋‹ค.์ œ 1 ์žฅ ์„œ ๋ก  1 ์ œ 2 ์žฅ ์‹คํ—˜ ๊ฒฐ๊ณผ 8 ์ œ 1 ์ ˆ ๋‹จ๋ฐฑ์งˆ ๋””์ž์ธ 8 ์ œ 2 ์ ˆ UV-Vis ์ŠคํŽ™ํŠธ๋Ÿผ ์ธก์ • 11 ์ œ 3 ์ ˆ Native PAGE ๋ถ„์„ 13 ์ œ 4 ์ ˆ Blue native PAGE ๋ถ„์„ 17 ์ œ 5 ์ ˆ ํ˜•๊ด‘ (Fluorescence) ๋ถ„์„ 21 ์ œ 6 ์ ˆ Mutant library ์ œ์ž‘ 25 ์ œ 7 ์ ˆ Mutant ๋‹จ๋ฐฑ์งˆ์˜ ๊ด‘ํ•™์  ํŠน์„ฑ ๋ถ„์„ 29 ์ œ 3 ์žฅ ๊ฒฐ๋ก  ๋ฐ ๊ณ ์ฐฐ 37 ์ œ 4 ์žฅ ์‹คํ—˜ ๋ฐฉ๋ฒ• 42 ์ œ 1 ์ ˆ ํด๋กœ๋‹ 42 ์ œ 2 ์ ˆ ๋‹จ๋ฐฑ์งˆ ๋ฐœํ˜„๊ณผ ์ •์ œ 43 ์ œ 3 ์ ˆ Native gel ๋ถ„์„ 44 ์ œ 4 ์ ˆ Blue native gel ๋ถ„์„ 44 ์ œ 5 ์ ˆ UV-Vis ์ธก์ • 45 ์ œ 6 ์ ˆ ํ˜•๊ด‘ (Fluorescence) ์ธก์ • 45 ์ œ 7 ์ ˆ Mutant library ํ•ฉ์„ฑ 46 ์ œ 8 ์ ˆ Mutant library screening 47 ์ฐธ๊ณ ๋ฌธํ—Œ 48 Appendix 53 Abstract 55Maste
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