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Samenvatting - toegepaste microbiologie theorie
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samenvatting en vertaling van H1-H5 van toegepaste microbiologie
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InhoudToegepaste microbiologie......................................................................................................................1
1. Genen.................................................................................................................................................1
1.1 Centrale dogma:...........................................................................................................................1
1.2 DNA replicatie...............................................................................................................................3
2. Regulatie van genexpressie................................................................................................................6
2.1 Transcriptie initiatie......................................................................................................................6
2.1.1 Induceerbare vs. onderdrukbare genen................................................................................6
2.1.2 Situatie 1: effect van een repressor op induceerbare genen.................................................7
2.1.3 Situatie 2: effect van een repressor op onderdrukbare genen..............................................7
2.1.4 Situatie 3: effect van een activator op induceerbaar gen......................................................8
2.1.5 Situatie 4: effect van een activator op een onderdrukbaar gen.............................................8
2.2 Transcriptie elongatie...................................................................................................................8
2.2.1 Attenuatie..............................................................................................................................8
2.2.2 Riboswitches........................................................................................................................10
2.3 Translatie....................................................................................................................................10
2.3.1 Kleine RNA moleculen..........................................................................................................10
2.4 Posttranslatie (niet in deze cursus).............................................................................................10
3. Genetische variatie...........................................................................................................................11
3.1 Tautomerisatie............................................................................................................................11
3.2 Soorten mutatie..........................................................................................................................12
3.3 Gevolg van mutaties...................................................................................................................12
3.4 Mutant detectie systeem............................................................................................................13
3.4.1 Replica plating:....................................................................................................................13
3.4.2 Mutant selectie:...................................................................................................................13
3.4.3 Ames test.............................................................................................................................14
3.5 DNA herstel systemen................................................................................................................15
3.5.1 Proofreading........................................................................................................................15
3.5.2 Excision repair (knippen).....................................................................................................15
3.5.3 Direct herstel.......................................................................................................................15
3.5.4 Mismatch herstel.................................................................................................................16
3.5.5 Recombinanten herstel........................................................................................................17
3.5.6 SOS response.......................................................................................................................17
3.6 DNA uitwisseling.........................................................................................................................18
3.6.1 Recombinatie op moleculair niveau.....................................................................................19
1
, 3.6.2 Bacteriële plasmiden...........................................................................................................22
3.7 Bacteriële transformatie.............................................................................................................23
3.8 Transductie.................................................................................................................................23
3.9 Het genoom achterhalen d.m.v. onderbroken paar proces........................................................24
4. Recombinant-DNA-technologie........................................................................................................25
4.1 Termen.......................................................................................................................................25
4.2 Organismen gebruikt in recombinatie........................................................................................25
4.3 Probleem bij recombinanten......................................................................................................25
4.4 Restrictie enzymen.....................................................................................................................25
4.5 Southern blotting........................................................................................................................26
4.6 cDNA = complementary DNA......................................................................................................27
4.7 Kloneren.....................................................................................................................................28
4.8 Kloneringsvector.........................................................................................................................29
4.8.1 Plasmiden als vector............................................................................................................30
4.8.2 fagen als vectoren................................................................................................................33
4.8.3 cosmids als vectoren............................................................................................................33
4.8.4 artificiële chromosomen als vectoren..................................................................................35
4.9 Expressievectoren.......................................................................................................................36
4.10 Mogelijke promotors................................................................................................................37
4.10.1 Lac-operon.........................................................................................................................37
4.10.2 Arabinose operon PBAD.......................................................................................................37
4.11 Wat wordt er gevormd bij expressievectoren..........................................................................37
4.12 Voorbeeld expressievector oligohistidine-expressievector (pTrcHis) ......................................38
4.13 Eukaryoten als expressiesystemen...........................................................................................39
4.14 Genetische manipulatie van planten........................................................................................39
4.15 Hoe zoek je een bepaald gen....................................................................................................40
4.16 Fenotypische rescue.................................................................................................................40
4.17 Gebruik van probe....................................................................................................................41
4.17.1 Verschil tussen probes & primers......................................................................................41
4.17.2 Genenbank.........................................................................................................................41
4.18 Genetic engineering..................................................................................................................41
4.18.1 Knock-outs.........................................................................................................................41
4.18.2 Reportergenen...................................................................................................................44
4.18.3 RNA-interferentie (RNAi) = gene silencing.........................................................................44
4.19 PCR...........................................................................................................................................45
4.19.1 Reverse PCR.......................................................................................................................45
2
, 4.19.2 Real time PCR.....................................................................................................................45
5. Genomics..........................................................................................................................................46
5.1 Sanger method...........................................................................................................................46
5.2 Next generation sequensing (NGS).............................................................................................47
3
, Toegepaste microbiologie
1. Genen
1.1 Centrale dogma:
Transcriptie: complementaire RNA streng o.b.v. DNA
Translatie: tRNA brengt aminozuur o.b.v. mRNA in de ribosoom
1
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