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    Title: 啤酒酵母菌之電穿孔高效率轉型及其在酵母菌雙雜合篩選之應用
    Other Titles: High-efficient transformation of saccharomyces cerevisiae by electroporation: application to yeast two-hybrid screening
    Authors: 林昕瑩;Lin, Hsin-ying
    Contributors: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    Keywords: 啤酒酵母菌;電穿孔法;酵母菌雙雜合篩選;electroporation;yeast two-hybrid screening
    Date: 2007
    Issue Date: 2010-01-11 02:30:08 (UTC+8)
    Abstract: 在第一部份是關於電穿孔法高效率轉型,由六個不同的基因YJR129C、YHR209W、EBNA2、shy A、NBS1及KPNA2去探討一些會影響轉型效率的變因,像是在培養基的製備、轉入質體DNA的預處理及轉入不同質量的質體DNA等。之後把高效率電穿孔法應用在酵母菌雙雜合篩選上,目的是藉由此法使酵母菌雙雜合的轉型達到更理想的效率,以利於由基因庫中,轉型的細胞數目能夠涵蓋完整的基因組。此步驟效率已達到2.5 x 106 /μg DNA。
    第二部分應用高效率電穿孔法於酵母菌的雙雜合篩選,以「魚、餌」的概念,由已知的基因YJR129C、YHR209W、EBNA2接上BD載體,做為「餌」,YJR129C、YHR209W各為啤酒酵母菌(Saccharomyces cerevisiae) BY4742上的一段基因,為假設性甲基轉移酶。EBNA2為EB病毒感染細胞後最先表現的基因之一,也是促使B細胞不死化的重要因子;再由已接上AD載體之未知目標基因之基因庫,為「魚」。送入啤酒酵母菌PJ69-4A中,有兩兩有結合的蛋白質就會在篩選的特定培養基中表現出來。之後,我們可以再把未知的目標基因做進一步的功能性分析,可以瞭解到各個蛋白之功能與特性。目前在EBNA2的酵母菌雙雜合篩選實驗中,所篩選出菌落數為421株。
    In part I, high efficiency transformation by electroporation is developed. Factors such as medium preparation, pretreatment of plasmid, quantities of plasmid, are investigated with six different genes YJR129C, YHR209W, EBNA2, shy A, NBS1 and KPNA2. The purpose of our protocol is to achieve ideal efficiency so that the number of transformants can saturate the library used in two-hybrid screening. This protocol has reached a efficiency of more than 2.5 x 106 /μg DNA.
    In part II, high efficiency transformation by electroporation is applied to yeast two-hybrid screening. Using a “fish-bait” concept, known genes YJR129C, YHR209W, EBNA2 are ligated to the BD vector as “the bait”. YJR129C and YHR209W are from Saccharomyces cerevisiae BY4742, both a putative methyltransferase gene. EBNA2 is one of the early expressed genes during EB virus infection, important in promoting the B cell immortalization. Appropriate DNA library constructed in the AD vector was screened for “the fish” when the library was transformed into yeast, PJ69-4A. The interacting proteins expressed from the library would be selected in the specific medium. Afterwards, more functional analysis may be done to understand role of the genes being selected and the characteristics of the protein product. In the EBNA2-interacting protein using the yeast two-hybrid screening, 421 positive clones were obtained.
    Appears in Collections:[Graduate Institue of Life Sciences] Thesis

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