Inhoud
Toegepaste 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
,Vernieuwde versie (gevolg van reverse DNA transcriptie en RNA replicatie door virussen):
DNA: structuur? Nucleotiden
Verschil DNA // RNA
Dubbele streng (ds)// enkele streng (ss)
ATGC // AUGC
Desoxyribose suiker // ribose suiker
Suiker + base: nucleoside
Nucleoside + fosfor: nucleotide
DNA en RNA worden gerepliceerd van 5’ naar 3’
Sanger sequencing: https://www.youtube.com/watch?v=9QPt8vQ5OZ4&t=25s
2
,1.2 DNA replicatie
Semiconservatief: moederstreng plus aansluiting van complementaire basen
Bij meesten prokaryoten bi-directioneel: één vork langs elke kant van de ORI-site
Rolling circle principe (bij bacteriële plasmiden of virale genomen):
DNA replicatie kan niet beginnen zonder primer
3
, DNA polymerase: Gamma complex in de replicatievork, beide core enzymen repliceren elk een streng
DNA, rode cirkel zorgt dat DNA open blijft, paarse bollen breekt waterstofbruggen waardoor
replicatievork open gaat
Leading en lagging strand, gevolg van 5’ 3’ replicatie
3’ naar 5’ van de moederstreng
Replicatie stopt op ter-site (terminatie-site)
A bindt met T/U
C bindt met G
4
Toegepaste 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
,Vernieuwde versie (gevolg van reverse DNA transcriptie en RNA replicatie door virussen):
DNA: structuur? Nucleotiden
Verschil DNA // RNA
Dubbele streng (ds)// enkele streng (ss)
ATGC // AUGC
Desoxyribose suiker // ribose suiker
Suiker + base: nucleoside
Nucleoside + fosfor: nucleotide
DNA en RNA worden gerepliceerd van 5’ naar 3’
Sanger sequencing: https://www.youtube.com/watch?v=9QPt8vQ5OZ4&t=25s
2
,1.2 DNA replicatie
Semiconservatief: moederstreng plus aansluiting van complementaire basen
Bij meesten prokaryoten bi-directioneel: één vork langs elke kant van de ORI-site
Rolling circle principe (bij bacteriële plasmiden of virale genomen):
DNA replicatie kan niet beginnen zonder primer
3
, DNA polymerase: Gamma complex in de replicatievork, beide core enzymen repliceren elk een streng
DNA, rode cirkel zorgt dat DNA open blijft, paarse bollen breekt waterstofbruggen waardoor
replicatievork open gaat
Leading en lagging strand, gevolg van 5’ 3’ replicatie
3’ naar 5’ van de moederstreng
Replicatie stopt op ter-site (terminatie-site)
A bindt met T/U
C bindt met G
4
