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Role of peroxide sensing transcription regulator OxyR in Streptomyces coelicolor : 방선균 Streptomyces coelicolor에서 OxyR 전사인자의 역할
DC Field | Value | Language |
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dc.contributor.advisor | 노정혜 | - |
dc.contributor.author | 네르민 악두만 | - |
dc.date.accessioned | 2017-07-19T09:05:58Z | - |
dc.date.available | 2017-07-19T09:05:58Z | - |
dc.date.issued | 2014-02 | - |
dc.identifier.other | 000000017255 | - |
dc.identifier.uri | https://hdl.handle.net/10371/131560 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 노정혜. | - |
dc.description.abstract | Abstract
OxyR is an H2O2 sensing transcriptional regulator of the LysR-family that is generally found in Gram-negative bacteria and some Gram-positive bacteria. In Streptomyces coelicolor, OxyR positively regulates its own gene and ahpCD encoding alkyl hydro peroxidase. Another peroxide-sensing regulator CatR controls catalase A production in response to H2O2. It binds to catA and catR genes and represses their transcription. Physiological study showed that ΔoxyR mutant grew faster in liquid YEME (complex medium) and NMMP (minimal medium). On solid media, ΔoxyR mutant exhibited better growth and sporulation even when media contained various concentrations of H2O2. It was postulated that catA expression may be elevated in ΔoxyR mutant to allow better growth phenotype. The amount of catalase A was determined by activity staining and was found to be higher in the mutant. This suggests that compensatory catalase induction may have conferred ΔoxyR with the better growth phenotype. Moreover, antibiotic actinorhodin was produced more in ΔoxyR in both solid SFM and R2YE media and liquid minimal medium. To investigate the regulatory role of OxyR and CatR, their target genes need be unraveled on genome scale, possibly by chromatin immunoprecipitation (ChIP). As an initial step to perform ChIP analysis, tagging of OxyR and CatR with detectable probe was performed. Cloning of 6xMyc tag to the C-terminal end of OxyR and CatR were done. Specific detection of tagged proteins by anti-Myc antibody was successful. Further improvement of detection sensitivity, either by changing the tag or solubility optimization, will be useful for genome-wide detection of direct binding sites of OxyR and CatR. Key words: Streptomyces coelicolor, OxyR, catalase, CatR, myc tagging, oxidative stress, actinorhodin | - |
dc.description.tableofcontents | Contents
국문초록................................................................................................................i Abstract.................................................................................................................ii Contents................................................................................................................iii List of figures........................................................................................................v List of tables.........................................................................................................vi List of abbreviations............................................................................................vii CHAPTER 1. Backgrounds...................................................................................1 1.1 Biology of Streptomyces coelicolor................................................................2 1.2 Oxidative stress responses...............................................................................2 1.2.1 Reactive oxygen species.........................................................................3 1.2.2 Biological defence systems to oxidative stress......................................5 1.3 Hydrogen peroxide sensing transcription regulators .....................................5 1.3.1 OxyR.......................................................................................................6 1.3.2 CatR........................................................................................................7 1.4 Catalases and peroxidases of Streptomyces coelicolor..................................9 1.4.1 catalases..................................................................................................9 1.4.2 Peroxidases.............................................................................................9 1.5 Aims of this study..........................................................................................10 CHAPTER 2. Material and Methods...................................................................11 2.1 Bacterial strains and culture conditions.........................................................12 2.2 General recombinant DNA techniques..........................................................16 2.2.1 Transformation of E.coli......................................................................16 2.2.2 Conjugation between E.coli and Streptomyces coelicolor...................16 2.2.3 PCR-based tandem epitope tagging system for Streptomyces coelicolor genome...............................................................................................16 iv 2.3 DNA analysis.................................................................................................17 2.3.1 Polymerase chain reaction (PCR)..........................................................17 2.3.2 DNA sequencing..................................................................................17 2.4 Protein analysis..............................................................................................21 2.4.1 Preparation of cell extracts...................................................................21 2.4.2 Western blot analysis............................................................................21 2.4.3 Catalase activity staining......................................................................21 2.4.4 Quantification of actinorhodin (Act) and undecylprodigiosin (Red)...22 CHAPTER 3. Results..........................................................................................23 3.1 Physiological growth of ΔoxyR in comparison with wild type......................24 3.1.1 Growth comparison in liquid media.....................................................24 3.1.2 Growth comparison on solid media......................................................24 3.1.3 Catalase A production in ΔoxyR mutant...............................................30 3.1.4 Antibiotic production in ΔoxyR mutant................................................32 3.2 PCR-based tandem epitope tagging for CHIP...............................................35 3.3.1 6xmyc tagging of oxyR in Streptomyces coelicolor.............................36 3.3.2 6xmyc tagging of catR in Streptomyces coelicolor..............................40 CHAPTER4.Discussion......................................................................................44 References...........................................................................................................49 | - |
dc.format | application/pdf | - |
dc.format.extent | 4458990 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Streptomyces coelicolor | - |
dc.subject.ddc | 570 | - |
dc.title | Role of peroxide sensing transcription regulator OxyR in Streptomyces coelicolor | - |
dc.title.alternative | 방선균 Streptomyces coelicolor에서 OxyR 전사인자의 역할 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Nermin Akduman | - |
dc.description.degree | Master | - |
dc.citation.pages | 63 | - |
dc.contributor.affiliation | 자연과학대학 생명과학부 | - |
dc.date.awarded | 2014-02 | - |
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