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研究生:劉文瑞
研究生(外文):Liu, Wen-Rui
論文名稱:大腸桿菌乳糖運輸酵素分析
論文名稱(外文):Analysis of permease mutant in escherichia coli
指導教授:程樹德程樹德引用關係
指導教授(外文):Tsen, Suh-Der
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
中文關鍵詞:微生物學免疫學大腸桿菌乳糖運輸素
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
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在自然的環境下,微生物族群已有些突變存在,這些突變發生在DNA層次上,經過轉錄(transcription)及轉譯(ranslation)後所產生的蛋白質之特性與未突變的不一定完全相同。然而這些突變之間大部份對細菌而言是有害的,因此在整個族群後續的繁衍下會被逐漸淘汰殆盡。只有非常微量的突變是有益的。然而在自然狀態,必須經過許多代的競爭及選擇,此突變才能成為優勢的菌種。
本實驗室前期學長賴思佳及龐中培的研究以lac operon為題材,研究細菌在特殊環境壓力下是否會使得較合適的突變種出現。因此在一個連續培養槽中(chemostat)(圖一)培養大腸桿菌,以乳糖為唯一碳源,當作是環境壓力(selection stress),而細菌體內首先面對此壓力的為乳糖運輸酵素,因其負責將乳糖帶到細菌體內。所以只要有較佳之乳糖運輸酵素存在時,經過多代的繁衍競爭後,此個體會取代原先的族群而成為主要的族群。
已知乳糖運輸酵素(lactose permease)為乳糖操作子(lacoperon)中之一員,是一個橫跨細胞膜的厭水性蛋白質(transmembrane hydrophobic protein)(圖二),它利用細胞膜間的質子化學位能差(proton chemical gradient)來催化運輸乳糖進入細菌體內(ref.19,20,21);乳糖運輸酵素之417胺基酸中有大約70%為厭水性,含有12個穿越細胞膜的α-heliX片段。而其N端和C端位於細胞質面。
為了加速適合的(fit)突變種出現,我在連續培養槽中培養大腸桿菌,每天加入突變劑(EMS)到連續培養槽中,經過15天的處理,將菌液塗在培養基上,選擇大的菌落,分別測試生長速率及乳糖運輸酵素對ONPG的Km值,進一步將乳糖運輸酵素基因選殖出來定序,以找出生長達率較快及乳糖運輸酵素Km值不同的細菌,看是否起因於乳糖運輸酵素DNA上的變化,才能更進一步研究其蛋白質上的改變和其酵素動力學上的特質。
In the natural environment, there are mutations in each microbial population. These mutations occur at the DNA level, and proteins that are produced from mutated genes may not have the same properties as unchanged ones. Most of these mutations, however, may be deleterious to the organism. As the population grow, these mutants would be selected against gradually. Only a very small fraction of these mutations may be beneficial. Many generations would be required for them to become predominant by natural selection.
Our lab has used lac operon as a model system to study whether fitter mutants can reach a higher frequency under environmental stress. So previous experiments used lactose as the only carbon source. Since lactose limitation became an important environmental stress, the lactose permease, which is responsible for transporting lactose from outside into cytoplasm, may be the prime target for selection. If a fitter mutant occurs in the population, it will replace the original population through many generations.
Lactose permease is a member of the lac operon. It is a trans-membrane hydrophobic protein. It uses energy from the trans-membrane proton electrochemical gradient to transport the lactose. About 70% of permease (417 residue) is hydrophobic and contains 12 transmembraneα-helix segments, and its N and C termini are exposed to cytoplasm.
In order to accelerate the generation of fitter mutants, I added mutagen (EMS) to the chemostat. After 15 days, the bacterial culture was spread on LB plates to select for larger colonies. The growth rate of cell from larger colonies were measured. The Km of lactose permease from faster-growing cells was also measured. The lactose permease gene were then cloned and sequenced to find whether differences in growth rate and Km are due to changes in DNA level. We can then investigate changes in enzyme activity.

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