臺灣博碩士論文加值系統

通過細菌與細菌之間的相互交換信息的流程稱爲群體感應(Quorum-sensing)，然而這流程能調諧部分基因但只限於一些菌類。東方弧菌是一種熒光細菌來自于中国北部黄河，具有六組基因相連著群體感應， 例如luxP (VIA_001151), luxQ (VIA_001152), luxS (VIA_003963), luxO (VIA_003573) 與 (VIA_003054), luxU (VIA_003574) 和 hapR (VIA_000273)。這項研究之所以專注於hapR基因因爲它與LuxR基因是同源體，歷來報告表示LuxR是哈維弧菌熒光操縱組（lux operon）的活躍剂。哈維弧菌與費希爾弧菌的LuxR基因并非是同源體因爲用來操控基因表現的主要群體感應調控組來自於哈維弧菌。
近來的研究觀察關於HapR的作用在於東方弧菌所發出的生物冷光（bioluminescence）。爲了確認HapR蛋白質與lux operon啓動子的相互作用，本研究采用管内，體内以及计算机模拟的進行。位於pET28a vector内, HapR基因的片段大小為615 bp 而它的蛋白質片段大小為24kDa，pET29a内的蛋白質為19kDa，然而融合蛋白質衹限於pET28a-HapR。給予HapR最佳IPTG引導為1 mM IPTG培養于溫度25 oC， 3小時，引用 Tris-HCl buffer pH8進行蛋白質純化。
管内研究的結果證實HapR與lux operon no.1-4啓動子有著相互作用而lux operon no.5 和6 卻沒有，直到上游lux operon no. 5 (292 bp) 啓動子才能足夠與HapR蛋白質結合。體内研究結果顯示HapR-PM結合體與ITPG為引導擁有最高熒光值(Relative Light Unit）的表現，例如HapR-PM2 在IPTG引導和5小時的培養后達到24.563 x 106 RLUs。以計算式的研究和分析，東方弧菌CIP 102891 = ATCC 33934 strain CIP 102891具有七組lux operon基因，luxCDABEGH，其排列方式與哈維弧菌ATCC 33843 (392 [MAV])頗爲相識，高達97.8%相識度。與此同時，東方弧菌僅僅依靠一種QS系統，或稱爲AI2系統用於掌控熒光度表現的作用。通過本系統能預測到冷光的調控機制依賴著磷酸化與無磷酸化的级联效应相似于哈維弧菌與費希爾弧菌的AI2系統。總結來説，對於東方弧菌熒光基因的調控，HapR都有著息息相關的角色。

The process of communication from one cell to another in bacteria called Quorum Sensing (QS). This process can regulate the genes that must be perform in a group of bacteria. In V. orientalis, the luminous bacteria from Yellow coast of China there are six genes that linked with QS. That genes were: luxP (VIA_001151), luxQ (VIA_001152), luxS (VIA_003963), luxO (VIA_003573) and (VIA_003054), luxU (VIA_003574) and hapR (VIA_000273). This research focused on hapR gene since it was LuxR homologues and previous report showed that LuxR is an activator of the lux operon in V. harveyi (Swartzman et al., 1992; Showalter et al.,1990). LuxR in V. harveyi is not homolog with the luxR in A. fischeri, since it was the QS master regulator that controls expression of the genes in the QS of V. harveyi.
This present study observed the role of HapR in bioluminescence process of V. orientalis. For confirmation, the interaction between HapR protein and promoter lux operon, the in vitro, in vivo and in silico study were done. DNA size of hapR was 615 bp and the protein size was about 24 kDa in pET28a, and around 19 kDa in pET29a. But only the pET28a that harbouring HapR that was protein fusion. The best treatment of IPTG induction for HapR was 1 mM IPTG in 25oC for 3 hour, and the buffer for purification the protein was Tris-HCl pH 8.
In vitro study proved that HapR has an interaction with the promoter lux operon no.1-4 but not with the promoter lux operon no.5 and 6. It means upstream of the promoter lux operon no. 5 (292 bp) are sufficient to bind with HapR protein. In vivo study result showed that clones that has the higest Relative Light Unit (RLUs) was in HapR-PM with IPTG induction group, which is HapR-PM2 with 24.563 x 106 RLUs at 5 hour after IPTG induction. This result suggest that HapR bind the promoter lux operon around 792 bp-581 bp. In silico study result suggest that V. orientalis CIP 102891 = ATCC 33934 strain CIP 102891 has seven lux operon genes, luxCDABEGH and the arrangement is similar to V. harveyi strain ATCC 33843 (392 [MAV]) with 97.8% similarities. In silico study also demonstrated that V. orientalis only use one type of QS system that was AI2 system to manage their light production and it predicted that the regulation of bioluminescence was depend on phosphorylation-dephosphorylation cascade similar with AI2 system in V. harveyi and A. fischeri. So, it’s can conclude that HapR has a role on the regulation of lux genes from V. orientalis.

中文摘要 I
ABSTRACT III
ACKNOWLEDEMENT V
TABLE OF CONTENT VI
LIST OF FIGURES IX
LIST OF TABLES XI
CHAPTER I. INTRODUCTION 1
1.1 Background 1
1.2 Purpose of study 3
CHAPTER II. LITERATURE REVIEW 4
2.1 Quorum sensing 4
2.1.1 Quorum sensing systems 5
2.1.1.1 Quorum sensing in gram negative bacteria: LuxI/LuxR-type 5
2.1.1.2 Quorum sensing in gram positive bacteria: peptide mediated 9
2.2 Vibrio orientalis 16
2.3 HapR 17
2.4 Lux operon 18
CHAPTER III. MATERIALS AND METHODS 20
3.1 Framework 20
3.2 Bacterial strain 20
3.3 Vector 20
3.4 Competent cell DH5α and BL21(DE3) 21
3.5 Bacterial medium 21
3.5.1 Luria-Bertani (LB) 21
3.5.2 Tryptic Soy Broth (TSB) 22
3.6 Genomic DNA 22
3.7 Molecular cloning techniques 23
3.7.1 Polymerase Chain Reaction (PCR) 23
3.7.2 Analysis of the PCR (agarose gel electrophoresis) 24
3.7.3 Ligation 25
3.7.4 Gel purification 25
3.7.5 Transformation 26
3.7.6 Plasmid extraction 27
3.7.7 Restriction analysis 27
3.8 Sequence analysis 28
3.9 Expression and purification of HapR 28
3.9.1 Protein expression 28
3.9.2 Protein purification 30
3.10 SDS-PAGE 31
3.11 In vitro study: Electrophoretic Mobility Shift Assay (EMSA) 32
3.11.1 Prepare the control and sample 33
3.11.2 Non-denaturing polyacrylamide gel electrophoresis 34
3.11.3 Staining DNA using SYBR green EMSA 34
3.12 In vivo study: bioluminescence intensity 35
3.13 In silico study 35
CHAPTER IV. RESULT 37
4.1 Genomic DNA extraction 37
4.2 Molecular cloning result 37
4.3 Analysis Sequence 39
4.4 Expression and purification of HapR 40
4.4.1 Protein expression 40
4.4.2 Purification of HapR 44
4.5 In vivo study: Electrophoretic Mobility Shift Assay (EMSA) 46
4.6 In vitro study: bioluminescence intensity 49
4.6.1 Relative Light Unit 49
4.6.2 Optical Density 52
4.7 In silico study: the comparison of lux gene in luminous bacteria 54
CHAPTER V. DISCUSSION 60
CHAPTER VI. CONCLUSION 74
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