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Targeting Lsr2/DNA complexation for dysregulation of gene expression in Mycobacterium tuberculosis
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables
List of Figures
List of Equations
List of Abbreviations
List of Symbols
1 Introduction
1.1 Tuberculosis: current status
1.1.1 Brief introduction to tuberculosis and its infectious process
1.1.2 Development of treatment regimens
1.1.2.1 The BCG vaccine
1.1.2.2 First line drugs
1.1.2.3 The issue of drug resistance and co-infection with HIV
1.2 Roles of Lsr2 in Mycobacterium tuberculosis
1.2.1 Lsr2: histone-like protein
1.2.2 Lsr2: global transcriptional regulator
1.2.3 Lsr2: physical barrier against ROIs
1.2.4 Current structural knowledge about Lsr2
1.3 Project goals
2 General Methods: Principles
2.1 Introduction
2.2 From genes to proteins
2.2.1 Polymerase chain reaction
2.2.2 Agarose gel electrophoresis
2.2.3 Restriction digest
2.2.4 Ligation
2.2.5 Transformation of competent cloning cells: plasmid replication
2.2.6 Plasmid purification
2.2.7 Transformation of competent expression cells
2.2.8 Induction of protein expression
2.2.9 Cell harvesting and lysis
2.3 Protein purification
2.3.1 Fast protein liquid chromatography (FPLC)
2.3.1.1 Immobilized metal affinity liquid chromatography
2.3.1.2 Ion exchange chromatography
2.3.2 Protein dialysis
2.3.3 SDS-PAGE: characterization of protein purity
2.3.4 Determination of protein concentration
2.4 Binding and inhibition studies on the protein of interest
2.4.1 Fluorescence Polarization assay
2.4.2 Intensity-fading MALDI mass spectrometry
2.5 Tests for growth inhibition of bacteria
2.5.1 Kirby-Bauer disk diffusion assay
2.5.2 Cell viability MTT colorimetric assay
2.5.3 Tests for bactericidal activity of a drug
2.6 Measuring gene expression: reverse transcriptase PCR
2.7 Protein crystallization strategy
3 Materials and Methods
3.1 Molecular cloning, expression and purification of Lsr2
3.2 Identifying potential inhibitors of Lsr2 DNA-binding function
3.2.1 Determination of Lsr2/DNA
3.2.2 Screening of NIH Clinical Collection
3.2.3 Tests on Zafirlukast derivatives and other potential inhibitors
3.3 Studies involving Zafirlukast
3.3.1 Determination of
3.2.2 Kirby-Bauer disk diffusion assays
3.3.3 Tests of the bactericidal activity of Zafirlukast
3.3.4 Determination of the minimal inhibitory concentration (MIC)
3.3.5 Semi-quantitative reverse transcriptase PCR
3.3.6 Intensity-Fading MALDI mass spectrometry assay
3.4 Molecular cloning, expression and purification of fusion proteins
3.4.1 Lysozyme-Lsr2
3.4.2 Lyso-Lsr2C
3.4.3 Lsr2N-T4-54-73-Lsr2C
3.5 Crystallization tray setup
3.5.1 Fusion proteins
3.5.2 Lsr2/DNA complexes
4 Zafirlukast is an Inhibitor of the Lsr2/DNA Complexation and Mycobacterial Growth
4.1 Expression and purification of Lsr2
4.2 Lsr2-DNA complexation
4.3 Zafirlukast inhibits Lsr2-DNA complexation
4.4 FP experiments performed on Zafirlukast derivatives
4.5 Zafirlukast directly binds Lsr2
4.6 Zafirlukast inhibits mycobacterial growth
4.7 Determination of Zafirlukast MIC
4.8 Zafirlukast exhibits bactericidal activity
4.9 Zafirlukast dysregulates gene transcription
5 Attempts at Lsr2 Crystallization
5.1 Synthesis of fusion proteins
5.1.1 Expression and purification of Lysozyme-Lsr2
5.1.2 Cloning of Lyso-Lsr2C
5.1.3 Expression and purification of Lsr2N-T4-54-73-Lsr2C
5.2 DNA design to crystallize the Lsr2-DNA complex
6 Conclusions and Future Work
References
A. lsr2 and Lsr2 sequences Bài viết liên quan
