臺灣博碩士論文加值系統

在台灣，有四株克雷伯氏肺炎菌(NTUH-K2044, NK8, NK29, NK245)分別在過去利用散彈槍定序法完成全基因體定序。傳統散彈槍定序的過程中，必須先將數倍的染色體DNA破碎成重疊的片段後並轉殖到質體 library進行放大，隨後進行Sanger定序，最後根據讀取的序列片段間重疊涵蓋的部分進行全基因體序列的組裝。一般來說，由於一些普遍存在基因體中的重複序列，電腦組裝往往會因為重複序列讀取片段的錯誤組裝而形成有間隔的數個組裝片段(contigs)。這種組裝序列的片段數目(或者說間隔的數目)對全基因體定序工作的完成度是很重要的指標。然而除了重複序列之外，我們認為在library製備的過程中，倘若有些DNA片段因為某些原因而無法轉殖成功，這些基因體片段可能也會導致序列組裝時產生破碎的現象。
為了驗證這些組裝問題的產生是不是因為基因體序列抑制了clone library的生長，我們分析了四株克雷伯氏肺炎菌散彈槍定序的數據，找出Consed組裝之中全部的間隔片段並且利用NCBI的BLAST功能映對在完成組裝與注解的基因體序列上。剔除掉與已知tRNA及rRNA基因座臨近的部分，其餘的間隔片段再分別依據他們之間的相關距離去歸納，間隔片段間距離小於1kb的歸類成一個共通間隔區域(common gap region; CGR)。我們根據基因注解從14個CGR中挑出29個基因及1個sRNA轉殖到受阿拉伯糖調控的可誘導表現載體上，並在大腸桿菌中誘導表現。藉由分析誘導表現之生長曲線來驗證這些基因片段對大腸桿菌生長可能的抑制。
實驗結果證實，27個成功完成基因轉植的菌株中，有16個菌株在進行阿拉伯糖誘導後或是沒有誘導的時候，生長曲線會被改變，依照影響的結果分為三類：(1)加入阿拉伯糖誘導後，生長曲線有抑制。(2)不論有沒有阿拉伯糖誘導，生長曲線都有抑制或提高。(3)實驗結果無法重複。在16個生長曲線會受到影響的大腸桿菌基因轉殖株中，有兩株分別是帶有sulA和minC的基因，而這兩個基因已經證實與細菌分裂有關。此外，殖入sulA與minC會對生長曲線有影響之外，我們也從顯微鏡中觀察到在阿拉伯糖誘導表現後會導致大腸桿菌外型改變。然而轉入克雷伯氏肺炎菌後，卻無此現象發生。
本篇研究結果驗證了有些無法組裝的間隔片段是由於DNA序列抑制clone library的生長而產生，也從中找尋出會影響細菌生長以及改變外型的重要生長調控基因。然而這些克雷伯氏肺炎菌的基因產物是如何影響大腸桿菌的生理，進而抑制了大腸桿菌的生長或形態，仍有待進一步的分析與研究。

Four K. pneumoniae strains, NTUH-K2044, NK8, NK29, NK245, have been sequenced respectively by whole genome shotgun approach in Taiwan. In the process of traditional shotgun sequencing, multiple copies of the genomic DNA must be sheared into overlapping fragments, ligated to plasmid vectors and transformed into E. coli. After that, the clone library is sequenced by Sanger method and the resulting reads were assembled computationally based on overlapping information. In a shotgun approach, reads containing the repeat sequences of the genome are usually misassembled and this causes gaps between contigs. While gaps were supposed to be eliminated during the finishing phase of the genome project, we consider these might be useful information for functional genomics discovery. By analyzing the gap sequences, it is possible to identify those so-called ‘non-cloneable sequences’ in the genome. These sequences are inhibitory to the growth of e E. coli transformants and thus are likely to be missing during shotgun sequencing approach.
To identify whether the genomic sequences inhibit the growth of E. coli, we analyzed the shotgun data of the four K. pneumoniae strains to find out all of the gaps in the initial Consed assembly and located the positions of gaps on each of the finished genomes. Gaps near to known repeats, such as the tRNA and rRNA sequences were excluded. Gaps with a physical distance within 1kb are grouped together into a common gap region (CGR). According to the gene annotation, we picked 30 candidate genes, including 29 protein-coding genes and 1 sRNA gene from 14 CGRs. The candidate sequences are PCR amplified and cloned into the arabinose-inducible expression vector and expression induced in E. coli. The inhibitory effects of these sequences were analyzed by monitoring the growth curve of the E. coli harboring each of the constructs.
The results showed that 16 of the 27 candidate sequences have altered the growth curve. The sequences are classified into 3 types: (1) Induction of candidate gene expression inhibits growth. (2) Candidate gene altered the growth curve, regardless of arabinose induction. (3) Non-conclusive, the results are difficult to repeat. Among the 16 influential constructions, we have identified two cell division associated genes, sulA and minC, that not only altered the growth curve of E. coli but also the cell morphology.
By analyzing the shotgun assembly gaps we have identified several gap regions shared among the four K. pneumoniae. Our analyses have identified several genes that might have an effect on the growth of bacteria. The effects of K. pneumoniae genes on E. coli physiology are interesting and further investigations are needed to understand the molecular mechanism of their targets.

Abstract I
摘要 III
Index V
Table Index VIII
Figure Index IX
Chapter 1. Introduction 1
1.1 Klebsiella pneumoniae in human infections 1
1.2 Four K. pneumoniae strains 1
1.3 Studies associated with K. pneumoniae genome projects 2
1.3.1 Impacts on studying antimicrobial resistance determinants 2
1.3.2 Metabolic diversity 3
1.3.3 Capsular polysaccharide diversity 3
1.3.4 Virulence genomic island 3
1.4 Sequencing of the four K. pneumoniae genomes 5
1.5 Conventional shotgun strategy using Sanger reads and its limitations 5
Chapter 2. Material and Method 8
2.1 Material 8
2.1.1 Shotgun data 8
2.1.2 Genomic DNA sequences 8
2.1.3 Programs 8
2.1.4 Bacterial strains 8
2.1.5 Culture medium 8
2.1.6 Plasmids 9
2.1.7 Primers 9
2.1.8 Reagents 9
2.2 Method 10
2.2.1 Mapping and analyses of shotgun gaps 10
2.2.2 Polymerase chain reaction (PCR) 11
2.2.3 Constructions of the inducible expression clones 12
2.2.4 Heat shock 14
2.2.5 Growth curve assay 14
2.2.6 Electroporation 15
Chapter 3. Result 17
3.1 Identification and mapping of common gap region (CGR) in the genomic shotgun assemblies 17
3.2 Construction of the candidate sequence clones 18
3.3 Effect of the CGR sequences on E. coli growth curve 19
3.3.1 Type I effect: an inhibitory effect in log phase after induction 20
3.3.2 Type II effect: an altered growth curve no matter arabinose was added or not 20
3.3.3 Type III effect: result was diddicult to repeat 21
3.4 Genes from the CGRs that altered the morphology of E. coli 21
3.5 Expression of SulA or MinC in K. pneumoniae 22
Chapter 4. Discussion 23
4.1 Analysis of the shotgun data 23
4.2 Uncloneable candidate genes from CGRs 24
4.3 Candidate sequence affected the growth curve without induction 24
4.4 Two cell-division associated genes 26
Chapter 5. Conclusion 28
Reference 60 

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