金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：143

、訪客IP：3.135.207.201

姓名

涂文峰(Wen-Feng Tu) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試
(Metallothionein expression in Escherichia coli and metal accumulation ability test)

相關論文

★ 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現並藉由microRNAs調控DNA修復機制	★ 研究Dicrotophos對HepG2細胞毒性之分子機制：CSA蛋白質在毒性扮演之角色
★ TNT經由ROS介導之內質網壓力及粒線體失衡誘導人類肝臟細胞凋亡	★ Pseudomonas sp. A46全基因組分析與重金屬復育基因工程菌開發
★ 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷	★ 三硝基甲苯之毒理機制及生物降解暨多氯乙烯汙染模場生物整治
★ 探討人類肝癌細胞HepG2經4-氨基聯苯處理過後miRNA-630對於同源重組修復相關蛋白MCM8的調控機制	★ 假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討
★ 開發新穎性包埋Dehalococcoides mccartyi及Clostridium butyricum之長效脫氯膠體	★ 探討DNA損傷反應與慢性暴露4-胺基聯苯產生之肝臟毒性
★ 以Lpp-OmpA工法建構新穎性基因工程菌強化鎘生物復育能力	★ 建構脫鹵球菌與固氮菌共培養系統促進氮源缺乏環境下的還原脫氯作用
★ 硒代胱氨酸通過誘導人肝細胞癌中的 DNA 損傷和抑制 DNA 修復途徑來增強順鉑敏感性	★ 轉錄體分析 Acetobacterium woodii 降解1,1,1-三氯乙烷機制並用以協助 Dehalococcoides進行還原脫氯
★ 以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY 降解二氯甲烷機制	★ 研究雙特松對HepG2細胞之DNA修復的影響

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

在於自然環境中，具有強烈的毒性，易造成人體危害。當生物暴露在重金屬環境時，為處理體內的重金屬會表現金屬硫蛋白，並與重金屬結合降低重金屬的毒性。根據前人文獻，建構並且比較3種形式的持續表現金屬硫蛋白(metallothionein, MT)載體在大腸桿菌中的表現情形，在大腸桿菌中只有單獨表現金屬硫蛋白的情況，因金屬硫蛋白是不穩定的小分子蛋白，因此在大腸桿菌中的表達並不是很穩定；然而當金屬硫蛋白接上綠螢光蛋白(EGFP)或是谷胱甘肽S-轉移酶(GST)時，金屬硫蛋白能在大腸桿菌中穩定表達。而在測試重金屬抗性，確定大腸桿菌能生長於150 ppm Ni2+ 和 Cd2+之培養基。因為大腸桿菌的鎳離子外排系統，使鎳離子不能長時間存在於大腸桿菌內，無法應用在鎳的生物復育，然而在大腸桿菌表達金屬硫蛋白之融合蛋白的情況，在培養24小時後能穩定結合著鎳離子，避免鎳離子在大腸桿菌體內的大量流失，有效吸收鎳離子1.5 mg/g，顯示金屬硫蛋白對於增加鎳的吸收量是有效的。

摘要(英)

Heavy metals exist in the natural environment and have strong toxicity, which is easy to cause harm to the human body. When organisms are exposed to heavy metals, metallothioneins combine with heavy metals to reduce the toxicity of heavy metals. The three types of sustained expression of metallothionein (MT) plasmid in Escherichia coli were constructed and compared. In Escherichia coli, only metallothionein was expressed alone, because metallothionein is a small molecule protein not stable, however, metallothionein is attached to green fluorescent protein (EGFP) or glutathione S-transferase (GST), metallothionein can be stably expressed in Escherichia coli. While testing for heavy metal resistance, it was confirmed that Escherichia coli can grow in medium of 150 ppm Ni2+ or Cd2+. Because of the nickel ion efflux system of Escherichia coli, nickel ions cannot be present in Escherichia coli for a long time, and cannot be used for bioremediation of nickel. However, the expression of metallothionein fusion protein in Escherichia coli, after 24 hours, to avoid the massive loss of nickel ions from Escherichia coli, and effectively absorb nickel ions 1.5 mg/g, indicating that metallothionein is effective for increasing the absorption of nickel.

關鍵字(中)

★ 重金屬
★ 生物復育
★ 金屬硫蛋白
★ 基因工程

關鍵字(英)

★ heavy metals
★ biological remediation
★ metallothionein
★ genetically engineered bacteria

論文目次

中文摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VI
一、緒論 1
1.1 研究緣起 1
1.2 重金屬特性 2
1.2.1 「鎘」 2
1.2.2 「鎳」 2
1.3 重金屬生物復育 3
1.4 基因工程與生物整治 5
1.4.1 金屬硫蛋白(Metallothionein) 5
1.4.2 基因工程菌與生物復育 5
二、研究動機與目的 7
三、實驗材料與方法 8
3.1 實驗材料 8
3.1.1 使用儀器與廠牌 8
3.1.2 常用藥品與試劑 9
3.2 實驗方法 13
3.2.1 核酸聚合反應(PCR) 13
3.2.2 DAN 純化 14
3.2.3 DNA電泳 14
3.2.4 限制酵素處裡(Digestion) 15
3.2.5 DNA膠體純化 15
3.2.6 DNA接合反應 (Ligation) 16
3.2.7 大腸桿菌電穿孔勝任細胞製備 16
3.2.8 大腸桿菌熱休克勝任細胞製備 17
3.2.9 大腸桿菌電穿孔轉型法 18
3.2.10 大腸桿菌熱休克轉型法 19
3.2.11 表現金屬硫蛋白之質體建構 20
3.2.12 質體萃取傳統法 20
3.2.13 質體萃取試劑組 21
3.2.14 蛋白質萃取(超音波震盪法) 22
3.2.15 蛋白質萃取(法式細胞破碎儀) 23
3.2.16 蛋白質濃度測定(Bradford protein assay) 24
3.2.17 蛋白質電泳 (SDS Poly-acrylamide gel electrophoresis) 24
3.2.18 蛋白質膠體染色 (Coomassie Brilliant Blue R染色法) 25
3.2.19 蛋白質純化 (IMAC-Ni column純化) 26
3.2.20 生長曲線 28
3.2.21 重金屬生長曲線分析 28
3.2.22 重金屬生物累積實驗 29
3.2.23 菌種保存與繼代 29
3.2.24 重金屬分析 30
四、實驗結果 32
4.1 持續表現MT蛋白載體pJBM之建構 32
4.2 持續表現MT-EGFP融合蛋白載體pJBME2之建構 32
4.3 持續表現MT-EGFP融合蛋白載體pJBME2之建構 32
4.4 Escherichia coli BL21大腸桿菌持續表現MT分析 33
4.5 Escherichia coli BL21大腸桿菌持續表現MT-EGFP融合蛋白分析 33
4.6 Escherichia coli BL21大腸桿菌持續表現GST-MT融合蛋白分析 33
4.7 轉殖基因Escherichia coli BL21大腸桿菌之生長曲線 34
4.8 轉殖基因Escherichia coli BL21大腸桿菌在75 ppm Ni2+之生長曲線 34
4.9 轉殖基因Escherichia coli BL21大腸桿菌在150 ppm Ni2+之生長曲線 34
4.10 轉殖基因Escherichia coli BL21大腸桿菌在200 ppm Ni2+之生長曲線 35
4.11 轉殖基因Escherichia coli BL21大腸桿菌在75 ppm Cd2+之生長曲線 35
4.12 轉殖基因Escherichia coli BL21大腸桿菌在150 ppm Cd2+之生長曲線 36
4.13 轉殖基因Escherichia coli BL21大腸桿菌在200 ppm Cd2+之生長曲線 36
4.14 轉殖基因Escherichia coli BL21大腸桿菌在250 ppm Cd2+之生長曲線 36
4.15 Escherichia coli BL21與基因工程菌重金屬生物固定分析 37
4.16 MT2-EGFP蛋白質純化分析 37
五、討論 38
5.1 MT以及融合蛋白之表現 38
5.2 表現MT以及融合蛋白影響Escherichia coli BL21生長速率分析 38
5.3 重金屬對於轉殖基因工程菌生長影響分析分析 39
5.4 Escherichia coli BL21與基因工程菌重金屬生物固定分析 40
結論 42
圖表 43
參考文獻 54
附表 60
附圖 61

參考文獻

1. P. B. Tchounwou et al. (2012) “Heavy Metals Toxicity and the Environment.” Experientia supplementum. 101: 133–164.
2. P. Chandra et al., (2004) “Chromium accumulation and toxicity in aquatic vascular plants.” The Botanical Review 70 (3): 313-327.
3. K. Yamamoto et al. (1993) “Kinetics and modelling of hexavalent chromium reduction in Enterobacter cloacae”. Biotechnology and Bioengineering. 41 129–133.
4. J. Jeyasingh et al. (2005) “Bioremediation of chromium contaminated soil: optimization of operating parameters under laboratory conditions.” Journal of Hazardous Materials B118 113–120
5. G. A. Atlanta (1999) Toxicological profile for mercury. Health Service Department of Health and Human Services.
6. S. Jensen et al. (1996) “Biological methylation of mercury in aquatic organisms.” Nature. 223: 753-754.
7. W. H. Xu et al. (2009) “Speciation of chromium in soil inoculated with Cr(VI)-reducing strain, Bacillus sp. XW-4” Journal of Central South University of Technology. Volume 16, Issue 2, pp 253–257
8. S. Das et al. (2016) “Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants” Applied Microbiology Biotechnology 100:2967–2984
9. E. Ebtesam et al. (2013) “Bioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteria.” Applied Water Science 3:181–192
10. H. Guo et al. (2010) “Bioremediation of heavy metals by growing hyperaccumulaor endophytic bacterium Bacillus sp. L14.” Bioresource Technology 101 8599–8605
11. S. Silver et al. (1984) “Bacterial transformations of and resistances to heavy metals.” Genetic Control of Environmental Pollutants Springer USA, pp. 23–46
12. J. T. Trevors et al. (1986) “Cadmium transport, resistance and toxicity in bacteria, algae, and fungi.” Canadian Journal of Microbiology 32:447–464
13. C. L. Hansen (1984) “Bacterial removal of mercury from sewage.” Biotechnology and Bioengineering. 26: 1330-1333
14. J. S. Chang et al. (1998) “Development of microbial mercury detoxification processes using mercury-hyperresistant strain of Pseudomonas aeruginosa PU21.” Biotechnology and Bioengineering. 57: 464-470
15. W. W. Zhang et al. (2012) “Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction.” Applied Microbiology Biotechnology. 93:1305–1314.
16. D. H. Nies et al. (1998) “CHR, a novel family of prokaryotic proton motive force-driven transporters probably containing chromate/sulfate antiporters.” Journal of Bacteriology 180:5799–5802
17. R. Batool et al. (2012) “Hexavalent chromium reduction by bacteria from tannery effluent.” Journal of Microbiology Biotechnology 22:547–554
18. C. Viti et al. (2013) “Molecular mechanisms of Cr (VI) resistance in bacteria and fungi.” FEMS microbiology reviews 38:633–659
19. R. Mishra et al. (2012) “Reduction of chromium-VI by chromium resistant Lactobacilli: a prospective bacterium for bioremediation.” Toxicology International 19:25–30
20. M. Margoshes et al. (1957). “A cadmium protein from equine kidney cortex." Journal of the American Chemical Society 79 (17): 4813–4814.
21. S. J. Felizola et al. (2014) “Metallothionein-3 (MT-3) in the human adrenal cortex and its disorders”. Endocrine Pathology. 25 (3): 229–235.
22. D. H. Hamer (1986) “Metallothionein.” Annual Review of Biochemistry 55:913-51.
23. Y. Liu (1995) “Transgenic mice that overexpress metallothionein-1 are protected from cadmium lethality and hepatotoxicity.” Toxicology and Applied Pharmacology. 135(2):222-8.
24. M. J. Stillman (1995) “Metallothioneins.” Coordination Chemistry Reviews. 144: 461-511.
25. T. Barac et al. (2004) “Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants.” Nature Biotechnology 22, 583–588.
26. O. N. Ruiz et al. (2011) “Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase” BMC Biotechnology 11:82
27. Y. J. Su et al. (2009) “Bioaccumulation of Arsenic in Recombinant Escherichia coli Expressing Human Metallothionein.” Biotechnology and Bioprocess Engineering 14: 565-570
28. X. Deng et al. (2013) “Comparative study on Ni2+-affinity transport of nickel/cobalt permeases (NiCoTs) and the potential of recombinant Escherichia coli for Ni2+ bioaccumulation.” Bioresource Technology 130 69–74
29. Xu Deng, et al. (2011) “Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake” Bioresource Technology 102 3083–3088
30. Z. Li et al. (2014) “Genes Conferring Copper Resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in Copper-Contaminated Soil.” Applied and Environmental Microbiology 03381-13
31. C. Viti et al. (2009) “Involvement of the oscA gene in the sulphur starvation response and in Cr(VI) resistance in Pseudomonas corrugata 28” Microbiology 155, 95–105
32. J. Xu et al. (2015) “Sources and remediation techniques for mercury contaminated soil” Environment International 42-53
33. T. Berka et al. (1988) “Efficient expression of the yeast metallothionein gene in Escherichia coli.” Journal of Bacteriology 170:21-26.
34. S. Chen1 et al. (1997) “Construction and Characterization of Escherichia coli Genetically Engineered for Bioremediation of Hg2+-Contaminated Environments” Applied and Environmental Microbiology 2442–2445
35. J. Chakraborty et al. (2014) “Characterization and cadmium-resistant gene expression of biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11.” Environmental Science and Pollution Research 21(24):14188-201.
36. T. Kimura et al. (2008) “Chromium(VI) inhibits mouse metallothionein-I gene transcription by preventing the zinc-dependent formation of an MTF-1-p300 complex” Journal of Biochemical 415(3)477-482
37. X. He et al. (2012) “Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium” Applied Microbiology and Biotechnology 95:1605–1613
38. D. C. Washington (2009) “Drinking Water Contaminants; United States.” Environmental Protection Agency (EPA): US EPA.
39. H. M. Salem et al. (2000) “Heavy metals in drinking water and their environmental impact on human health.” International Journal of Biosciences Cairo University: Giza, Egypt; pp. 542–556.
40. M. A. Khan et al. (2007) “Effect of environmental pollution on heavy metals content of Withania somnifera.” Journal of the Chinese Chemical Society 54, 339–343.
41. N. Das et al. (2008) “Biosorption of heavy metals” An overview. Indian Journal of Biotechnology 7, 159–169.
42. A. D. Chodak et al. (2008) “The impact of nickel on human health.” Journal of Elementology 13, 685–696.
43. W.C. Wang et al. (2014) “Characteristics, functions, and applications of metallothionein in aquatic vertebrates.” Frontiers in Marine Science Volume 1 Article 34 1
44. E. Carpene et al. (2007) “Metallothionein functions and structural characteristics.” Journal of Trace Elements in Medicine and Biology S1, 35–39
45. M. Valls et al. (2000) “Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil.” Nature Biotechnology volume18, pages661–665
46. R. Dixit et al. (2015) “Bioremediation of Heavy Metals from Soil and Aquatic Environment: An Overview of Principles and Criteria of Fundamental Processes.” Sustainability 7, 2189-2212
47. T. Berka et al. (1988) “Efficient Expression of the Yeast Metallothionein Gene in Escherichia coli.” Journal of Bacteriology, p. 21-26
48. S. L. Chen et al. (1998) “Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor.” Biotechnology Progress, 14:667-671.
49. F. Yang,et al. (2007) “High-yield expression in Escherichia coli of soluble human MT2A with native functions.” Protein Expression and Purification, Pages 186-194
50. I. Shachrai et al. (2010) “Cost of Unneeded Proteins in Escherichia coli Is Reduced after Several Generations in Exponential Growth.” Molecular cell Pages 758-767
51. C. J. Williams et al. (1998) “Comparison between biosorbents for the removal of metal ions from aqueous solutions.” Water Research 32 (1), 216–224
52. G. P. Williams et al. (2012) “Biosorption and bio-kinetic studies of halobacterial strains against Ni2+, Al3+ and Hg2+ metal ions.” Bioresource Technology. 107, 526–529.
53. C. J. CastilloZacarias et al. (2011) “Biosorption of metals by phenolresistant bacteria isolated from contaminated industrial effluents.” African journal of microbiology research. 5 (18), 2627–2631.
54. S. Ozdemir et al. (2012) “Cd, Cu, Ni, Mn and Zn resistance and bioaccumulation by thermophilic bacteria, Geobacillus toebiisubsp decanicus and Geobacillus thermoleovorans subsp stromboliensis.” World Journal of Microbiology and Biotechnology. 28 (1), 155–163.
L. S. Ferreira et al. (2011) “Adsorption of Ni2+, Zn2+ and Pb2+ onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris. I. Single metal systems.” The Chemical Engineering Journal 173 (2), 326–333.
55. Y. Ma et al. (2011) “Cd(II) and As(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins.” Journal of Hazardous Materials 185 (2011) 1605–1608
56. J. H. R. Kagi et al. (1988) “Biochemistry of metallothionein.” Biochemistry 27, 8509-8515.
57. Z. Khan et al. (2015) “Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium.” Applied Microbiology Biotechnology 99(24):10745–10757
58. X.Deng et.al. (2003) “Bioaccumulation of nickel from aqueous solutions by genetically engineered Escherichia coli.” Water Research 37(10):2505–2511
59. Y.M. Zhang et al. (2007) “Cloning and expression of the nickel/cobalt transferase gene in Escherichia coli BL21 and bioaccumulation of nickel ion by genetically engineered strain.” Huan Jing Ke Xue 28(4):918–923
60. A. Duprey et al. (2014) “NiCo Buster: engineering Escherichia coli for fast and efficient capture of cobalt and nickel.” Journal of Biological Engineering 8(1):1–11
61. F. N. Stahler et al. (2006) “The novel Helicobacter pylori cznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, ureasemodulation, and gastric colonization.” Infection and Immunity 74: 3845–3852
62. A. Rodrigue et al. (2005) “Identification of rcnA (yohM), a nickel and cobalt resistance gene in Escherichia coli.” Journal of Bacteriology 187:2912–2916

指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2019-7-11

推文