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General consideration about Gene Expression and expression studies

General consideration about Gene Expression and expression studies. Expression studies Expression Host -> Expression System Promoter system -> expression vector Properties of product -> stability Production level. Expression studies. 1. Analyzing Transcription - Northern blot

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General consideration about Gene Expression and expression studies

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  1. General consideration about Gene Expression and expression studies • Expression studies • Expression Host -> Expression System • Promoter system -> expression vector • Properties of product -> stability • Production level

  2. Expression studies 1. Analyzing Transcription - Northern blot - Micro array - real-time PCR - Primer extension 2. Promoter studies Use of report genes to study regulatory elements 3. Analyzing Translation - Western blot - immuno assays - 2D electrophoresis - proteomics

  3. Northern Blot -> to study transcription level

  4. Studying Transcription Microarray technique – DNA chips

  5. Studying Transcription Microarray technique – DNA chips

  6. Microarray technique to study upregulation and downregulation of gene expression

  7. Studying Transcription Primer Extension

  8. Promoter Studies • Used reporter genes: • Lac Z • GFP • Luciferase Promoter Reporter gene

  9. Promoter studies by using reporter genes

  10. Use of green fluorescent protein (GFP) as a reporter gene. Page 119

  11. Analyzing Translation – Western Blot

  12. 2 D Electrophoresis

  13. Comparison of expression systems

  14. Prokaryotic Expression vector

  15. Eukaryotic Expression vector

  16. -> mainly in prokaryotes (E. coli) -> 2µ plasmid in yeast -> some virus vectors in mammalien cells -> mainly in eukaryotes -> some bacterial systems (Bacillus)

  17. Gene Expression Transcriptional start Translational start

  18. Principal factors in bacterial expression

  19. Type of expression vectors

  20. PstI BamHI NarI NdeI SalI HindIII S/D phoA cutinase T7/lac Term phoA-cut pFCEX1 Prokaryotic Translational vector

  21. Gene Expression • Gene copy number: • 1. Plasmid copy number: • The copy-number of a plasmid in the cell is determined by regulating the initiation of plasmid replication. • The initiation of plasmid replication may be controlled by: • the amount of available primer (RNA) • the amount of essential replication proteins • the function of essential replication proteins. • 2. Gene dosage -> number of genes integrated into chromosome • - prokaryotic systems -> i.e. Transposons, phages, recombinantion • - mainly eukaryotic systems

  22. Incompatibility of plasmids: Not all plasmids are able to coexist in the same cell. Plasmids which have the same replication control functions are incompatible, and are assigned to the same incompatibility group (inc group). Plasmids of one incompatibility group are related to each other, but cannot survive together in the same bacterial cell, as only different kinds of plasmids are compatible. Ensures that we can make libraries -> just one plasmid taken up by one cell

  23. Homologous integration into chromosome Insertion on Bacillus subtilis chromosome

  24. mRNA stability

  25. Translation Initiation

  26. Codon Usage of Organisms Rare codons -> pauses translation -> lower production level

  27. Fusion proteins • increase production level • facilitate purification (taq) • detection of expression (GFP fusion) • Redirection of proteins (secretion -> signal peptidases) • Surface display (for screening of libraries) • Tandem arrays (for small peptides, toxic proteins,..)

  28. Some problems of production in E. coli

  29. Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell Protein Folding Page 116

  30. Some E.coli expression host considerations

  31. Secretion of Protein -> to avoid inclusion body formation at high expression level

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