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14 주차 수업자료

14 주차 수업자료. Chapter 19. Analysis of Gene Expression. Reporter gene: antibiotic resistance. Reporter gene: lacZ. lacZ encodes β -galactosidase Substrates Lactose ONPG X-gal. Reporter gene: phoA. phoA encodes alkaline phosphatase Substrates o -Nitrophenyl phosphate X-phos

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14 주차 수업자료

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  1. 14주차 수업자료

  2. Chapter 19. Analysis of Gene Expression

  3. Reporter gene: antibiotic resistance

  4. Reporter gene: lacZ • lacZ encodes β-galactosidase • Substrates • Lactose • ONPG • X-gal

  5. Reporter gene: phoA • phoA encodes alkaline phosphatase • Substrates • o-Nitrophenyl phosphate • X-phos • 4-Methylumbelliferyl phosphate

  6. Reporter gene: luc (or lux) • luc encodes luciferase (in firefly) • Substrates • Luciferin • luciferin + O2 + ATP  oxidized luciferin + CO2 + H2O + AMP + diphosphate + hν • Detect luminescence with luminometer

  7. Reporter gene: gfp • gfp encodes green fluorescent protein (GFP) • GFP: stable, non-toxic protein from jellyfish • Excitation at 395 nm, emission at 510 nm • GFP variants: eGFP, eYFP, eCFP, RFP, and etc. • Subcellular localization

  8. Gene fusion

  9. Deletion analysis of upstream region

  10. Gel retardation assay (electrophoretic mobility shift assay or bandshift assay) Labeling required

  11. DNA footprint analysis

  12. Primer extension assay & S1 nuclease assay: to identify the transcriptional start site

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