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研究生:鄭君瑜
論文名稱:NisA啟動子於枯草桿菌宿主中持續及調控表現特性
論文名稱(外文):Functional analysis of nisA promoter in bacillus subtilis under constitutive and controlled expression
指導教授:葉娟美
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:nisA啟動子枯草桿菌
相關次數:
  • 被引用被引用:1
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  • 下載下載:13
  • 收藏至我的研究室書目清單書目收藏:1
NisA 啟動子為NICE系統(nisin-controled expression system)元件之一。可經由nisin誘導其表現,此啟動子之表現係利用微生物之二元訊息傳導系統(two-component signal transduction system),由反應調節蛋白 NisR與感應蛋白NisK傳導nisin誘導因子之訊息,啟動PnisA或PnisF 啟動子下游基因的表現。
本研究以nisin誘導雙質體系統之nisA啟動子,來表現分泌胞外Bacillus subtilis WB800之鹼性彈性蛋白酶於時,研究發現,此系統之誘導效果雖不如預期,但它卻可持續大量表現鹼性彈性蛋白酶。為了了解nisA啟動子之特色,本研究亦構築了胞內表現系統,以葡萄醣醛酸酶(β-glucuronidase,GusA)為報告蛋白之胞內表現系統。在單質體系統由,GusA可以持續表現於枯草桿菌WB800細胞中,GusA之活性有兩個高峰,一為對數生長初期,一為產孢期,顯示nisA啟動子,可能可以被二種以上σ-RAN聚合酶所轉錄。在枯草桿菌DB104第二個表現高峰較不明顯,但表現量較高。而在本研究所構築之修飾雙質體系統調控表現情況下,Bacillus subtilis WB800中未發現酵素活性的表現,而在枯草桿菌DB104之研究中,顯示NisR、NisK在枯草桿菌宿主中扮演不同於乳酸菌中活化子之角色。
中文摘要 ……………………………………………………….…. i
英文摘要 …………………………………………………….……. ii
壹、前言 …………………………………………………….……. 1
貳、材料與方法 ………………………….………………………. 12
一、菌種與質體 ……………………………………………….... 12
二、藥品與試劑 ..………………………………………………… 14
三、質體抽取法 …..……………………………………………… 14
四、DNA分子之電泳、剪切、回收及黏合 ..………………….. 15
五、電轉形 ..……………………………………………………… 17
六、表現載體之構築 ..…………………………………………… 18
七、生長曲線測定 ……………………………………………… 19
八、鹼性彈性蛋白酶(Subtilisin YaB)活性測定 ..……………….. 19
九、葡萄醣醛酸酶(β-glucuronidase,GusA)活性測定………….. 20
參、結果與討論 ..………………………………………………… 21
一、載體之構築 ………………………..………………………… 21
二、分泌性蛋白質鹼性彈性蛋白酶(Subtilisin YaB)之表現 …… 25
三、胞內報導蛋白質葡萄醣醛酸酶(β-glucuronidase)之表現 …. 32
肆、結論 ………………………………………………………….. 49
伍、參考文獻 …………………………………………………….. 50
王志鵬,2001。一、枯草桿菌Bacillus subtilis DB104電轉形效率之增進。二、設計、合成第一型抗凍蛋白基因及其於枯草桿菌、大腸桿菌表現。國立中興大學食品科學系碩士論文。
李佩芳、邢禹依、周德源。2000。Promoter Activity of a Soybean Gene Encoding a Seed Maturation Protein, GmPM9。中央研究院植物學彙刊 41(3):175-182
曾夢蛟、蔡啟仁。 1994。 β-glucuronidase (GUS)基因轉移至馬鈴薯之研究。中國園藝 40(2): 142-160。
Airaksinen U, Penttila T, Wahlstrom E, Vuola JM, Puolakkainen M, Sarvas M. 2003. Production of Chlamydia pneumoniae proteins in Bacillus subtilis and their use in characterizing immune responses in the experimental infection model. Clin Diagn Lab Immunol. 10:367-75.
Birnboim, H. C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513.
Boylan, S. A., M. D. Thomas, and C. W. Price. 1991. Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor σB of Bacillus subtilis. J. Bacteriol. 173:7856-78806
Braaz R, Wong SL, Jendrossek D. 2002. Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis. FEMS Microbiol. Lett. 9:237-241.
Brückner, R. 1992. A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene. 122:187-192
Bryan, E. M., Bae, T., Kleerebezem, M. and Dunny, G. M. 2000. Improved vectors for nisin-controlled expression in gram-positive bacteria. Plasmid 44:183-190
Chan M-T, Chang H-H, Lo S-L, Tong, W-F, Yu S-M (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric β-amylase promoter /β-glucuronidase gene. Plant Mol. Biol. 22:491-506
Cohen, R., and Mire, M. 1971. Analytical-band centrifugation of an active enzyme-substrate complex. Determination of active units of various enzymes. Eur. J. Biochem. 23:276-81
Conrad, B., Savchenko, R. S., Breves, R., and Hofemeister, J. 1996. A T7 promoter-specific, inducible protein expression system for Bacillus subtilis. Mol. Gen. Genet. 250:230-236
Contaxis, C., and Reithel, F.1971. Studies on Protein Multimers. The Association-Dissociation Behaviour of beta-Galactosidase in Glycerol, Biochem. J. 124:623-627
de Ruyter P. G., Kuipers O. P., de Vos W. M. 1996. Controlled gene expression system for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ. Microbiol. 62:3662-3667
Débarbouillé, M., I. Martin-Verstraete, A. Klier, and G. Rapaport. 1991. The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both σ54 and phosphotransferasesystem-dependent regulators. Proc. Natl. Acad. Sci. USA 88:2212-2216.
Dwivedi C, Heck WJ, Downie AA, Larroya S, Webb TE. 1990. Effect of calcium glucarate on β-glucuronidase activity and glucarate content of certain vegetables and fruits. Biochem. Med. and Metab. Bio. 3:83-92.
Eichenbaum Z., Federle, M. J., Marra, D., de Vos, W. M., Kuiper, O. P., Kleerebezem, M. and Scott, J. R. 1998. Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comaprison of inductin level and promoter strength. Appl. Environ. Microbiol. 64:2763-2769
Engell, P. and Flock, J. I. 1992. The autocatalytic processing of the subtilisin Carlsberg pro- region is independent of the primary structure of the cleavage site. Mol. Microbiol. 6:1115-1119
Gunthert, U., Reiners, L. and Lauster, R. 1986. Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis. Gene. 41:261-70
Harwood, C. R. 1992 Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol. 10:247-256
Harwood, C. R. and Cutting, S. M. 1990. In molecular biological methods for Bacillus. pp. 392, John Wiley Press. New York, USA.
Helmann, J. D. 1991. Alternate sigma factors and the regulation of flagellar gene expression. Mol. Microbiol. 5:2875-2882.
Hoffmann, T., Toup, B., Szabo, A., Hungerer, C., and Jahn, D. 1995. The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. FEMS Microbiol. Lett. 131:219-225.
Horikoshi, K. and Teruhiko, A. 1982. Alkalophilic microorganisms. A new Microbial world. Japen scientific Soc. Press.
Horsburgh MJ, Thackray PD, Moir A. 2001. Transcriptional responses during outgrowth of Bacillus subtilis endospores. Microbiology 147: 2933-2941.
Kato Y, Asano Y. 2003. High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts. Protein Expr. Purif. 28:131-9.
Jang, J. S., Park, D. K., chun, M. and Byun, S. M. 1993. Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directied mutagenesis. Biochim. Biophys. Acta. 1162:233-235
Johnson, W.C., C. P. Moran, Jr., and R. Losick. 1983. Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene. Nature (London) 302:800-804
Kuipers, O. P., Beerthuyzen, M. M., deRuyter, P. G., Luesink, E. J., and deVos, W. M. 1995. Autoregulation of nisin biosynthesis in Lactococcus latis by signal transduction. J. Biol. Chem. 270:27299-304.
Kuipers, O. P., deRuyter., p. g., Kleerebezem, M., de Vos, W. M. 1998. Quorum sensing controlled gene expression in lactic acid bacteria. J. Biotechnol. 64:15-21.
LeGrice, S. F. 1990. Regulated promoter for high-level expression of heterologous genes in Bacillus subtilis, p. 201-214. In D. V. Goeddel (ed.), Gene expression technology. Academic Press, London, England.
Leighton, T. J. and Doi, R. H. 1971. The stability of messenger RNA during sporulation in B. subtilis. J. Biol. Chem 246:3189-3195.
McDonald, I. R., Riley, P.W., Sharp, R. J., and McCarthy, A. J. 1995. Factors affecting the electroporation of Bacillus subtilis. J. Appl. Bacteriol. 79:213-218.
Miller, J. F. 1994. Bacterial transformation by electroporation. Methods Enzymol. 235:373-385
Moran, C. P., Jr., N. Lang, S. F. J. LeGrice, G. Lee, M. Stevens. A. L. Sonenshein, J. Pero, and R. Losick. 1982. Nucleotide sequences that signal the initation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346.
Nagarajan, V., Albertson, H., Chen, M., and Ribbe, J. 1992. Modular expression and secretion vectors for Bacillus subtilis. Gene. 114:121-126
Nicholson, W. L., D. X. Sun, B. Setlow, and P. Setlow. 1989. Promoter specificity of sigma G-contaning RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters. J. Bacteriol. 171:2708-2718
Ohta Y, Inouye M. 1990. Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro. Mol. Microbiol. 4:295-304
Osburne, M. S. and Craig, R. J. 1986. Activity of two strong promoters cloned into Bacillus subtilis. J. Gen. Microbiol. 132:565-568
O''Sullivan, D. J., Walker S. A., West, S. G., Klaenhammer, T. R. 1999. Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression. Biotechology. 14:82-7
Platteeuw, C., Simons, G., de Vos, W. M. 1994. Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria. Appl. Environ. Microbiol. 60:587-93
Predich, M., G. Nair, and I. Smith. 1992. Bacillus subtilis early sporulation gene kinA, spo0F, and spo0A are transcribed b the RNA polymerase containing σH. J. Bacteriol. 174:2771-2778.
Roels, S., A. Driks, and R. Losick. 1992. Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis. J. Bacteriol. 174:575-585
Southward CM, Surette MG. 2002. The dynamic microbe: green fluorescent protein brings bacteria to light. Mol. Microbiol. 45:1191-6.
Sørenesen, K. I., Larsen, R., Kibenich, A., Junge, M. P. and Johanson, E. 2000. Afood-grade cloning system for industrial strains of Lactococcus lactis. Apple. Environ. Microbiol. 66:1253-1258
Steers, E., Craven, G., Anfinsen, C., and Bethune, J. 1965. Evidence for Non-Identical Chains in the beta-Galactosidase of Eschericia coli K12. J. Biol. Chem. 240, 2478-84
Sullivan, M. A., Yasbin, R. E., Young, F. E. 1984. New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene 29:21-26
Sun, D., P. Fajardo-Cavazos, M. D. Sussman, F. Tovar-Rojo, R.-M. Cabrera-Martinez, and P. Setlow. 1991. Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E-σF :identification of features of good E-σF-dependent promoters. J. Bacteriol. 173:7867-7874
Tasi, Y. C., Yamasaki, M., Yamamoto-Suzuki, Y. and Tamura, G. 1983. A new alkaline elastase of alkalophilic Bacillus. Biochem. International. 7:577-583
Tenu, J., Viratelle, O., and Yon, J. 1972. Kinetic Study of the Activation Process of beta-Galactosidase from Escherichia coli by Mg2+, Eur. J. Biochem. 26:112-118
Tenu, J., Viratelle, O., Garnier, J., and Yon, J. 1971. pH Dependence of the Activity of beta-Galactosidase from Escherichia coli, Can. J. Biochem. 20: 363
Vasantha, N. and Thompson, L. D. 1986. Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences. J. Bacteriol. 165:837-842
Wu, X. C., Lee, W., Tran, L., and Wong, S. L. 1991. Enginerring a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular protease. J. Bacteriol. 173:4952-4958
Zheng, L., Halberg, S. Roels, H. Ichikawa, L. Kroos, and R. Losick. 1992. Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes. J. Mol. Biol. 226:1037-1050.
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