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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
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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
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3 Northern Blot -> to study transcription level
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4 Studying Transcription Microarray technique – DNA chips
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5 Studying Transcription Microarray technique – DNA chips
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6 Microarray technique to study upregulation and downregulation of gene expression
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8 Studying Transcription Primer Extension
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9 Promoter Studies Used reporter genes: - Lac Z - GFP - Luciferase PromoterReporter gene
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10 Promoter studies by using reporter genes
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11 Use of green fluorescent protein (GFP) as a reporter gene. Page 119
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12 Analyzing Translation – Western Blot
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13 2 D Electrophoresis
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14 Comparison of expression systems
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15 Prokaryotic Expression vector
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16 Eukaryotic Expression vector
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17 -> mainly in prokaryotes (E. coli) -> 2µ plasmid in yeast -> some virus vectors in mammalien cells -> mainly in eukaryotes -> some bacterial systems (Bacillus)
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18 Gene Expression Transcriptional start Translational start
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19 Principal factors in bacterial expression
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20 Type of expression vectors
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21 Prokaryotic Translational vector PstI T7/la c phoAcutinase NarI SalI HindIII BamHI phoA-cut NdeI S/D Term pFCEX1
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22 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
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23 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
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24 Homologous integration into chromosome Insertion on Bacillus subtilis chromosome
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25 mRNA stability
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26 Translation Initiation
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27 Codon Usage of Organisms Rare codons -> pauses translation -> lower production level
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28 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,..)
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30 Some problems of production in E. coli
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31 Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell Page 116 Protein Folding
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32 Some E.coli expression host considerations
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33 Secretion of Protein -> to avoid inclusion body formation at high expression level
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