Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)Thiết kế vector biểu hiện con đường sinh tổng hợp provitamin A trong E. coli (Khóa luận tốt nghiệp)
E coli : Chính quy Chuyên ngành Khoa : CNSH - CNTP Khoá : 2011 2015 THÁI NGUYÊN - 2015 VITAMIN A TRONG E coli : Chính quy Chuyên ngành : : K43 - CNSH Khoa : CNSH - CNTP : 2011 2015 Khoa CNSH-CNTP THÁI NGUYÊN - 2015 i n, h Sinh viên L ii Trang 19 EcoRI HindIII 24 EcoRI XbaI 24 26 iii DANH M Trang 10 10 13 -crtY 15 -iEIB 16 16 ng vevtor pET28 21 27 -crt i EcoRI HindIII 29 -Y 30 hai Ezyme EcoRI HindIII 31 crtY vector pET-Y 32 crtY pET-Y 33 -iEIBY 34 Hình 4.7: K - XbaI EcoRI 35 -iEIBY 36 i Hình 4.10: B 37 pET- iEIBY E coli BL21 38 iv Bp : Base pair DNA : Deoxyribonucleic Acid E coli : Escherichia coli EDTA : Etilendiamin tetraaxetic acid IPTG : Isopropyl Thiogalactoside Kb : Kilo base LB : Laria Broth TAE : Tris-acetate-EDTA X-gal : 5-bromo-4-chloro-3-indoly- -D-galactoside v Trang PH N M 1.1 tv U 1.2 M c tiêu nghiên c u tài c c ti n PH N T NG QUAN NGHIÊN C U khoa h c c tài 2.1.1 Provitamin A vai trò c a provitamin A th c ti n ng t ng h p provitamin A 2.2 T ng quan nghiên c c 14 2.2.1 Tình hình nghiên c u v c 14 2.2.2 Tình hình nghiên c u v provitamin A th gi i 14 PH NG, N U 15 ng v t li u nghiên c u 15 ng nghiên c u 15 3.1.2 V t li u nghiên c u 15 3.1.3 Hóa ch t 17 3.1.4 D ng c thi t b nghiên c u 19 m th i gian nghiên c u 20 3.3 N i dung 20 3.3.1 N i dung 20 3.3.2 N i dung 20 u 20 vi PH N K T QU VÀ TH O LU N 28 4.1 idi-crtE-crtI-crtB-crt 28 4.1.1 K t qu g n crtY vào vector pET28 t o vector tái t h p pET-Y 28 4.1.2 K t qu g n c m gene iEIB (idi, crtE, crtI, crtB) vào vector pET-Y t o vector tái t h p pET-iEIBY 34 4.2 K t qu PH N K T LU u hi n c a pET- iEIBY E coli BL21 38 NGH 39 5.1 K t lu n 39 5.2 Ki n ngh 39 1.1 Provitamin A (ß- - p (Vilchez cs, 2011)[8] Ngoài p (Takahashi cs, 1998)[9] -caroten, ßcaroten, lycopene, lutein crytoxamthin (Rao and Rao, 2007)[10] Ngoài p ( 6)[4], pro (Paiva and Russell, 1999)[11] p (Lee cs, 2003)[12] provitamin (Johnson cs, 1996)[13] p p p ng - (Slattery cs, 2000) p p p provitamin A E coli p e idi, crtE, crtI, crtB crtY p 29 -crtY Hình 4.1: K I HindIII SM 0311 ) Y Y crt - pET28 30 crt g crt crt crt v -Y vào E coli nuôi - hình 4.2 Hình 4.2 òng mang vector pET-Y -> 6: dòng plasmid pET-Y pET28 pET28 d crtY vector vector pET28 crtY 31 d pET- EcoRI e HindIII Hình 4.3 K hai Ezym EcoRI HindIII pET28 -Y enzyme EcoRI HindIII (hai enzyme crtY vector pET28 crt 32 crtY chèn vào vector crtY vector pET28, crtY vector pET28 crtY pET-Y Hình 4.4: K Y vector pET-Y -Y entific -Y pETr pET-Y có Enzyme XbaI Enzyme EcoRI gene crt e 33 Hình 4.5 Y pET-Y crt EcoRI trí XbaI - e EcoRI XbaI 143 c crt EcoRI XbaI crt e crt gene crt -Y 34 4.1.2 iEIB (idi, crtE, crtI, crtB) vào vector pET-Y t -iEIBY iEIB E coli pET-iEIBY n Hình 4.6 dòng plasmid pET-iEIBY -Y -> 6: dòng plasmid - gene (iEIB pET- pET-iEIBY có 35 pET- XbaI EcoRI e iEIB ã -Y hay không iEIB e XbaI e idi) enym EcoRI enzyme crtB), pETe iEIB 6387 Hình 4.7 plasmid pET-iEIBY b I EcoRI pET-iEIB 36 6387 iEIB vector pET- iEIB pET- iEIBY pET-iEIBY - iEIBY 1768 bp 37 iEIB pET- iEIBY, e NcoI NotI iEIB Nco gene crt Not EIB iEIB NcoI NotI iEIB NcoI NotI 2370 bp, iEIB vector pET- iEIBY 38 - iEIBY 4.2 - iEIBY E coli BL21 ET- dòng E coli BL21 E coli BL21 pET- p Hình 4.10: B pET- iEIBY E coli BL21 E coli BL21 mang pET28 E coli BL21 mang pET-iEIBY iEIBY E coli BL21 39 pET- iEIBY cho provitamin A vi E coli E coli BL21 provitamin A iEIBY vector khác coli coli provitamin A coli -134(23) pp 140 (2003) Tinh - Tách ananatis lycopene Das, A., et al (2007) "An update on microbial carotenoid production: application of recent metabolic engineering tools." Appl Microbiol Biotechnol 77(3): 505-512 Vilchez, C., et al (2011) "Marine carotenoids: biological functions and commercial applications." Mar Drugs 9(3): 319-333 Takahashi, S., et al (1998) "A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis." Proc Natl Acad Sci U S A 95(17): 9879-9884 10 Rao, A V and L G Rao (2007) "Carotenoids and human health." Pharmacol Res 55(3): 207-216 11 Paiva, S A and R M Russell (1999) "Beta-carotene and other carotenoids as antioxidants." J Am Coll Nutr 18(5): 426-433 12 Lee, P C., et al (2003) "Biosynthesis of structurally novel carotenoids in Escherichia coli." Chem Biol 10(5): 453-462 13 Johnson, E A and W A Schroeder (1996) "Microbial carotenoids." Adv Biochem Eng Biotechnol 53: 119-178 14 Slattery, M L., et al (2000) "Carotenoids and colon cancer." Am J Clin Nutr 71(2): 575-582 15 Britton, G (1995) "Structure and properties of carotenoids in relation to function." FASEB J 9(15): 1551-1558 16 Debjani Dutta, U.R.C.(2005), Structure, health benefits, antioxidant property and processing and storage of carotenoids Department of Food Technology and Biochemical Engineering, Jadavper University, Kolkata- 700032, India: p 153-155 17 Bertram, J S (1999) "Carotenoids and gene regulation." Nutr Rev 57(6): 182-191 18 Misawa, N., et al (1991) "Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora." Appl Environ Microbiol 57(6): 1847-1849 19 Heller, F R., et al (1998) "LDL oxidation: therapeutic perspectives." Atherosclerosis 137 Suppl: S25-31 20 Palozza, P., et al (2008) "The protective role of carotenoids against 7-ketocholesterol formation in solution." Mol Cell Biochem 309(1-2): 61-68 21 Singh, N., et al (1995) "Oxidative stress and heart failure." Molecular and Cellular Biochemistry 147(1): 77-81 22 Dhalla, N S., et al (2000) "Role of oxidative stress in cardiovascular diseases." J Hypertens 18(6): 655-673 23 Reaven, P D., et al (1994) "Susceptibility of human LDL to oxidative modification Effects of variations in beta-carotene concentration and oxygen tension." Arterioscler Thromb 14(7): 1162-1169 24 Gordon, J E and M Schooff (2002) "Can high-dose supplementation with vitamins C and E, beta carotene, and zinc slow the progression of macular degeneration?" J Fam Pract 51(2): 105 25 Ausich, R L (1997) "Commercial opportunities for carotenoid production by biotechnology." Pure and Applied Chemistry 69(10): 2169 2174 26 Del Campo, J A., et al (2000) "Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp (Chlorophyta)." J Biotechnol 76(1): 51-59 27 Ernst, H (2002) "Recent advances in industrial carotenoid synthesis*." Pure Appl Chem 74(11): 2213 2226 28 Bailey, J (1991) "Toward a science of metabolic engineering." Science 252(5013): 1668-1675 29 Ikeda, M and R Katsumata (1992) "Metabolic Engineering To Produce Tyrosine or Phenylalanine in a Tryptophan-Producing Corynebacterium glutamicum Strain." Appl Environ Microbiol 58(3): 781-785 30 P Lee, C S.-D ( 2002) "Metabolic engineering towards biotechnological production of carotenoids in microorganisms." Appl Microbiol Biotechnol 60(1-2): 1-11 31.Yutaka Miura, K K., Hiroshi Shimada, Toshiko Saito, Katsumi Nakamura andNorihiko Misawa* (1998) "Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid." Biotechnology and Bioengineering 58(2-3): 306 308 32.Norihoko Misawa, M N., Kazuo Kobayashi, Shigeyuki Yamano, Yuko Izawa, Katsumi Nakamura, and Keij Harashima (1990) "Elucidation of Pathway the Erwinia uredovora Carotenoid Biosynthetic by Functional Analysis of Gene Products Expressed in Escherichia coli." JOURNAL OF BACTERIOLOGY 59(3): 6704-6712 33.Raman Babu, Natalia Palacios Rojas, Shibin Gao, Jianbing Yan and Kevin Pixley (2012) "Validation of the effects of molecular marker polymorphisms in LcyE and CrtRB1 on provitamin A concentrations for 26 tropical maize populations." International Journal of Plant Breeding Research: 122-012-1987-3 34 Monica A Schmidt1, Wayne A Parrott , David F Hildebrand , R Howard Berg , Amanda Cooksey, Ken Pendarvis , Yonghua He , Fiona McCarthy and Eliot M Herman (2014) "Transgenic soya bean seeds -carotene exhibit the collateral enhancements of oleate and protein content traits." Biotechnology Journa: pp 11 Plant Biotechnology JournaPlant ... : Base pair DNA : Deoxyribonucleic Acid E coli : Escherichia coli EDTA : Etilendiamin tetraaxetic acid IPTG : Isopropyl Thiogalactoside Kb : Kilo base LB : Laria Broth TAE : Tris-acetate-EDTA... 2002)[27] ProvitaminA 11 t carotenoid (Ausich, 1997)[25] (Bailey, 1991)[28] (Ikeda and Katsumata, 1992)[29] gi (P Lee, 2002) 12 có th (Yutaka Miura, 1998) qua a chúng (Norihiko Misawa, 1990)[32] coli. .. CaCl2 - Glycerol - Kháng sinh Ampiciline - Xgal 40 mM + IPTG 400 mM STT Scie plas Ltd Gel Logic 1500 Canada Bio Water system Pall Co.UK Nu F N Kodak USD 425 400E Nuaire USA 51 BW Horiba Japan