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標題: | 大腸桿菌中DNA拷貝數變異、外染色體DNA及非常規重組之發現 Discovery of copy number variation (CNV), extrachromosomal DNA (ecDNA), and illegitimate recombination events in E. coli |
作者: | 崔昭胤 Chao-Yin Tsuei |
指導教授: | 廖俊智 James C. Liao |
關鍵字: | 大腸桿菌,人工演化,拷貝數變異(CNV),外染色體DNA(ecDNA),細菌人工染色體(BAC),微染色體(miniC),合成生物學, Escherichia coli,Laboratory Evolution,Copy Number Variation (CNV),Extrachromosomal DNA (ecDNA),Bacterial Artificial Chromosome (BAC),Mini-Chromosome (miniC),Synthetic Biology, |
出版年 : | 2024 |
學位: | 博士 |
摘要: | 人工演化在研究生物進化及工業應用上扮演重要的角色。本論文研究大腸桿菌(E. coli)在篩選條件下的人工演化,探討了單步突變、拷貝數變異(CNV)、外染色體DNA(ecDNA)和微染色體(miniC)。研究首先分析大腸桿菌中的單步突變,通過將改造後含有提前終止密碼子之氯黴素乙醯轉移酶(cat)基因和RNA聚合酶亞單位β(rpoB)基因嵌入細菌人工染色體(BAC)中作為研究模型。實驗結果顯示了在cat基因提前終止密碼子之核苷酸單步突變模式,以及發現rpoB基因相較於在BAC上更傾向於在染色體中做單步突變的現象。此外,通過具有一段DNA拷貝數變異區段長度為7kb且含有完整ddp 操縱子的大腸桿菌中發現其拷貝數變異在人工演化中新穎特性,並且該區段中的ddp 操縱子為負責D-alanyl-D-alanine(DADA)之代謝。進一步分析顯示在染色體的雙向複製終止(EBR)區附近具有拷貝數變異發生率之增加,並證明了在串聯重複中發現的chi位點與拷貝數增加存在關聯。我們的研究結果亦指出拷貝數變異中的串聯重複可以外染色體DNA的形式存在,並且可由具有拷貝數變異的菌株中提取外染色體DNA並將其轉化至不同宿主,突顯其自主複制和利用同源重組整合到基因組中的特性。我們也識別到不含DNA複制起點的外染色體環狀DNA(eccDNA)之存在。此外,我們利用微染色體模擬了大腸桿菌中的拷貝數變異生成和非常規重組的過程,展示了微染色體在大腸桿菌進化中的實用性。總結來說,本研究不僅增進了我們對微生物基因組的理解,亦展示了拷貝數變異和非常規重組在細菌進化中的多樣功能,為合成生物學和人工演化領域提供了重要的發現和啟示。 Laboratory evolution utilizes many of the evolutionary strategies in biology and demonstrates its application in industry. This thesis investigates laboratory evolution of Escherichia coli under selective pressure, focusing on mechanisms including one-step mutations, copy number variations (CNV), extrachromosomal DNA (ecDNA), and mini-chromosome (miniC). The study started with an analysis of one-step mutations in E. coli using the chloramphenicol acetyltransferase (cat) gene interrupted with an early stop codon and RNA polymerase subunit β (rpoB) gene on Bacterial Artificial Chromosome (BAC). It quantitatively examines the mutation patterns for acquiring nucleotide change on cat with an early stop codon and reveals a bias towards mutations in genome-embedded rpoB compared to those on the BAC. Furthermore, we investigated the properties of an E. coli strain that exhibits significant CNV within a 7kb DNA region encompassing the entire ddp operon, crucial for D-alanyl-D-alanine (DADA) metabolism. Our analysis reveals an increased occurrence of CNV near the end of the bidirectional replication (EBR) region on the genome, and demonstrates the involvement of chi-site found in tandem repeats is linked to copy number increases. We further found that the tandem repeats within the CNV can exist as extrachromosomal DNA (ecDNA). We demonstrated that ecDNA can be extracted from strains with CNV and transformed into a different host. Surprisingly, the ecDNA can sustain in the new host also in the extrachromosomal form without an origin of replication. Alternatively, the ecDNA can sustain in the new host by integrating into the genome via homologous recombination or illegitimate recombination. The presence of extrachromosomal circular DNA (eccDNA) among ecDNA, lacking known DNA replication origins is identified as well. Additionally, we introduce a mini-chromosome model that simulates CNV generation and the illegitimate recombination processes, demonstrating its utility in the evolution of E. coli strain. In summary, this thesis enhances our understanding of microbial genome and reveals the functions of CNV, ecDNA, and illegitimate recombination in bacterial evolution, offering valuable insights for synthetic biology and laboratory evolution. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92381 |
DOI: | 10.6342/NTU202400675 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 生化科學研究所 |
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