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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 賴信志 | |
dc.contributor.author | Wei-Wen Tsai | en |
dc.contributor.author | 蔡維文 | zh_TW |
dc.date.accessioned | 2021-06-13T05:50:20Z | - |
dc.date.available | 2008-08-02 | |
dc.date.copyright | 2006-08-02 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-06 | |
dc.identifier.citation | Adler J and Templeton B (1967) The effect of environmental conditions on the motility of Escherichia coli. J Gen Microbiol, 46, 175-184.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33970 | - |
dc.description.abstract | Serratia marcescens是一種會產生紅色色素,隸屬腸道桿菌科 (Enterobacteriaceae)中靈菌屬 (Serratia)的革蘭氏陰性菌。其為一伺機性致病菌,易造成院內感染,而導致免疫低下的病人致病。Swarming為其細菌群體在固體培養基表面移行的一種特殊型態。S. marcescens 野生株CH-1在30℃時,具有在0.8% 培養基上swarming的現象,但在37℃的環境下,卻無swarming的表現型態。為進一步瞭解其中的機制,利用transposon mutagenesis (mini-Tn5-km1) 的方式,在37℃找到一株precocious-swarming mutant。對其進行研究發現,transposon嵌入的位置,在manA ORF上游約211 nucleotides處,此位置位於預測promoter region的範圍內。推測可能是由於此基因的表現量改變,造成37℃時的表現型與野生株有所差異。而目前已知manA製造出的蛋白質mannose-6-phosphate isomerase,其功能為轉換fructose-6-P (Fru-6-P) 及 mannose-6-P (Man-6-P),是連接能量代謝及mannose代謝的橋樑。為了進一步研究37℃時precocious-swarming的原因是否導因於manA基因的過量表現或者降低表現,我針對野生株CH-1進行了manA的基因剔除以及overexpression的實驗。取得突變株後,觀察其與CH-1在37℃時swarming的表現,推測manA的過量表現應為造成precocious-swarming的原因,且可能同時活化能量代謝及mannose代謝途徑而達成。 | zh_TW |
dc.description.abstract | Serratia marcescens is a red-pigmented gram-negative enteric organism. In addition, it is an opportunistic human pathogen and causes many nosocomial infections. Swarming is a specialized form of bacterial surface translocation when bacteria were cultured on surface of solid media. S. marcescens CH-1 swarms on 0.8% LB agar at 30℃, but not at 37℃. In order to understand the underlying mechanism of temperature-dependent regulation of swarming behavior, transposon mutagenesis was performed to screen for the mutants that swarm at 37℃. One precocious-swarming mutant (named WW100) was found and further experiments showed that the insertion site of transposon lay in the predicted promoter region of manA. The manA encodes a protein named mannose-6-phosphate isomerase whose function is interconverting fructose-6-phosphate and mannose-6-phosphate, and linking the glucose and mannose metabolic pathway. In order to investigate whether the phenomenon of WW100 precocious-swarming at 37℃ was due to increase or decrease in expression of manA gene, knockout and over-expression for manA were performed. The results here showed that precocious-swarming phenotype of S. marcescens WW100 was probably because of over-expression of manA, leading to activation of both energy metabolic and mannose metabolic pathways. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T05:50:20Z (GMT). No. of bitstreams: 1 ntu-95-R93424020-1.pdf: 1306392 bytes, checksum: 2ec338687260fb071e17ff53153e3d3f (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 中文摘要 1
Abstract 2 Chapter 1: Introduction 1.1 Serratia marcescens is an important opportunistic pathogen 3 1.2 Differentiation and multicellularity 3 1.3 Swarming behavior 3 1.4 Flagellum-dependent swarming motility 4 1.5 Biosurfactant production is essential for swarming motility 6 1.6 Biofilm, the city of microbes 6 1.7 manA transposon mutant, a precocious-swarming strain 9 1.8 Brief introduction of mannose-6-phosphate isomerase 9 1.9 Mannose metabolic pathway and synthesis of lipopolysaccharide in 11 Bacteria 1.10 The background for initiation of this thesis 15 1.11 Aims of this thesis 16 Chapter 2: Materials and methods 2.1 Bacterial strains, plasmids and primers 18 2.1.1 Bacteria strains 18 2.1.2 plasmids 18 2.1.3 primers 19 2.2 Materials and reagents 19 2.2.1 Kit 19 2.2.2 Medium 19 2.2.3 Antibiotics 20 2.2.4 Enzymes and chemicals 20 2.2.5 Electrophoresis reagents 21 2.2.6 Southern blot hybridization reagents 21 2.2.7 Plasmid DNA isolation reagents 22 2.2.8 Chromosomal DNA purification reagents 22 2.2.9 General chemicals 22 2.2.10 Miscellaneous 23 2.2.11 Equipment and Supplies 23 2.3 Experimental procedures 2.3.1 Isolation of plasmid DNA 25 2.3.2 Purification of chromosomal DNA 25 2.3.3 Extraction of DNA from agarose gel 26 2.3.4 Restriction enzyme digestion 26 2.3.5 Ligation reaction 26 2.3.6 Calcium chloride transformation 26 2.3.6.1 Preparation of transformation-competent cells 26 2.3.6.2 Transformation with DNA 26 2.3.7 Electroporation. 27 2.3.7.1 Preparation of electroporation-comptement cells 27 2.3.7.2 Electroporation of DNA 27 2.3.8 Blue-white screening for recombinant plasmids 27 2.3.9 Sequence analysis and databank comparison 28 2.3.10 Plasmid transfer from E. coli S17-1 to S. marcescens by conjugation 28 2.3.11 Southern Blot Hybridization 29 2.3.12 DIG-detection assay 29 2.3.13 Strategies used to knockout manA 29 2.3.14 Construction of recombinant plasmid for gene overexpression 30 2.3.15 Mobility assay 30 2.3.15.1 Swarming assay of S. marcescens CH-1 30 2.3.15.2 Swimming assay of S. marcescens CH-1 31 2.3.16 Measurement of bacterial growth 31 2.3.17 Quantitation of biofilm formation 31 Chapter 3 : Results 3.1 Characterization of the precocious-swarming phenotype of mini-Tn5 transposon mutant S. marcescens WW100. 32 3.2 Alignment of S. marcescens Man-6-P isomerase amino acid sequence with some Man-6-P isomerase homologues.32 3.3 Construction of manA insertion-deletion mutant S. marcescens WW101 derived from S. marcescens CH-1. 34 3.4 Characterization of WW101 phenotypes. 37 3.5 Mannose-6-phosphate isomerase links the fructose-6-phosphate and mannose-6-phosphate metabolism. 39 3.6 Mannose and glucose metabolic pathways both affect swarming phenotype. 40 3.7 Mannose-6-phosphate isomerase overproduction stimulates swarming at 37℃. 41 3.8 Biofilm formation is affected in WW100 but not in WW101 42 Chapter 4: Discussion 44 Index 49 Reference 55 Figure contents Fig. 1.1 Composition of the flhDC regulon. 5 Fig. 1.2 The biofilm formation of P. aeruginosa on an abiotic surface. 7 Fig. 1.3 The physical map of the 2.9 kb DNA fragment cloned from S. marcescens CH-1 chromosomal DNA. 9 Fig. 1.4 The PMI functions in carbohydrate metabolism. 10 Fig. 1.5 Structure and biosynthesis of mycobacterial mannose-containing glycoconjugates in M. tuberculosis cell wall. 12 Fig. 1.6 Hypothesized mannose metabolic pathway in Cryptococcus neoformans. 13 Fig. 1.7 Bacterial lipopolysaccharides. 14 Fig. 1.8 Pathway of specific O-antigen biosynthesis. 15 Fig. 1.9 Hypothesized mannose metabolic pathway in S. marcescens. 16 Fig. 3.1 Swarming motility of S. marcescens CH-1 and WW100. 32 Fig. 3.2 33 Fig. 3.3 Construction of the plasmid pWW101 carrying recombinant manA regions. 35 Fig. 3.4 PCR primers and the agarose gel electrophoresis of colony PCR products for preliminary screening of manA knockout mutant. 35 Fig. 3.5 Southern blot hybridization analyses for restriction map analysis. 36 Fig. 3.6 The growth dynamics of WW100, WW101 and CH-1 at 30℃ and 37℃. 37 Fig. 3.7 Swarming of CH-1, WW100 and WW101. 38 Fig. 3.8 The swimming motility of CH-1, WW100 and WW101. 39 Fig. 3.9 Survival assay of CH-1, WW100 and WW101 on various carbohydrate source plates. 40 Fig. 3.10 Swarming of CH-1, WW100 and WW101 on various carbohydrate source plates. 41 Fig. 3.11 manA gene in-trans over-expression in CH-1 and WW102 and swarming phenotype at 37℃. 42 Fig. 3.12 Quantification of biofilm formation. 43 Fig. 4.1 Hypothesis of role of manA in Serratia marcescens swarming behavior. 46 Fig. 4.2 Simplified scheme of the cytosolic and ER part of the N-glycosylation pathway. 47 Table 2.1 The bacteria strains used in this thesis. 18 Table 2.2 The plasmids used in this thesis. 18 Table 2.3 The primers used in this thesis. 19 | |
dc.language.iso | en | |
dc.title | Mannose-6-phosphate isomerase調控靈菌群體表面移行之多細胞行為 | zh_TW |
dc.title | Mannose-6-phosphate isomerase is involved in regulation of multi-cellular swarming behavior in Serratia marcescens | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 胡小婷,鄧麗珍,許萬枝,賈景山 | |
dc.subject.keyword | 靈菌,表面移行,多細胞行為, | zh_TW |
dc.subject.keyword | Serratia marcescens,swarming, | en |
dc.relation.page | 64 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-07 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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