Coordinated regulation of gene expression by E ... - Jacobs University

TABLE OF CONTENTS
2.2.5 Preparation of competent E.coli cells using calcium chloride ……….. 25
2.2.6 Transformation of E.coli cells ………………………………………… 25
2.2.7 Purification of nucleic acids by Phenol:Chloroform extraction ……… 26
2.2.8 Concentrating and quantification of nucleic acids …………………… 26
2.2.9 Polyacrylamide sequencing gels ……………………………………… 26
2.2.10 Growth curves and norfloxacin treatment ……………………………. 27
2.2.11 Extraction and quality check of total RNA …………………………… 27
2.2.12 Microarray hybridization …………………………………………….. 28
2.2.13 Microarray analysis …………………………………………………… 29
2.2.14 Real-time PCR ……………………………………………………….. 30
2.2.15 High resolution agarose gel electrophoresis and determination of sigma (σ)
……………………………………………………………………….. 30
2.2.16 In vitro transcription …………………………………………………. 31
2.2.17 Primer extension ……………………………………………………… 32
2.2.18 Potassium permanganate footpriniting ……………………………….. 32
2.2.19 β-galactosidase assay ………………………………………………… 33
3 Results ……………………………………………………………………….. 34
3.1 Growth phase dependent global transcriptional regulation ………… 34
3.1.1 Growth of CSH50 and LZ strains ……………………………………. 34
3.1.2 A novel microarray based approach to derive growth phase-specific
transcript profiles organized by supercoiling and chromatin proteins
………………………………………………………………………….. 36
3.1.3 Changes of supercoiling during growth ……………………………… 38
3.1.4 Genomic wheels of supercoiling sensitivity …………………………. 39
3.1.5 Variations of supercoiling-associated transcripts …………………….. 42
3.1.6 Supercoiling sensitivity of biosynthesis and degradation pathways …. 45
3.1.7 Coordination of metabolic pathways by supercoiling sensitivity …….. 45
3.2 Role of supercoiling and chromatin protein FIS in transcriptional
regulation of an individual promoter ………………………………… 49
3.2.1 Mechanism of transcriptional regulation by FIS & supercoiling: tyrT
promoter paradigm
…………….…………………………………………………………… 49
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