LABJOURNAAL
Moleculaire techniek
Het effect van TPR op PP5
Een onderzoek naar de functie van het regulatoire tetratricopeptide repeat (TPR) domein op de
fosfoatase enzym activiteit van het eiwit fosfatase 5 (PP5) in Rattus norvegicus (RnPP5)
Femke te Dorsthorst
Studentennummer: 13120107
,Inhoud
Gene editing..........................................................................................................................................5
Titel....................................................................................................................................................5
Onderzoeksvraag...............................................................................................................................5
Hypothese..........................................................................................................................................5
Doel...................................................................................................................................................5
Voorspellingen...................................................................................................................................5
Agarose gel elektroforese..............................................................................................................5
DNA-purificatie..............................................................................................................................5
Golden Gate Assembly...................................................................................................................6
Materiaal en Methode.......................................................................................................................6
PCR.................................................................................................................................................6
Agarose gel elektroforese..............................................................................................................7
DNA-purificatie & DNA concentratie meting.................................................................................7
Golden Gate Assembly...................................................................................................................7
Logboek.............................................................................................................................................8
PCR:................................................................................................................................................8
Agarose gel elektroforese:.............................................................................................................8
Golden Gate Assembly:..................................................................................................................9
Resultaten........................................................................................................................................10
Elektroforese:..............................................................................................................................10
DNA-purificatie............................................................................................................................10
Conclusie/Discussie.........................................................................................................................10
Transformatie......................................................................................................................................11
Titel..................................................................................................................................................11
Onderzoeksvraag.............................................................................................................................11
Hypothese........................................................................................................................................11
Doel.................................................................................................................................................11
Voorspellingen.................................................................................................................................11
Transformeer E.coli......................................................................................................................11
Plate en incubate.........................................................................................................................11
Colony PCR & Agarose gel electrophorese...................................................................................12
Mini-prep plasmid DNA isolation.................................................................................................12
DNA digestie................................................................................................................................12
Sanger sequence..........................................................................................................................13
, Materiaal en Methode.....................................................................................................................13
Transformeer E. coli BL21............................................................................................................14
Plate en incuberen.......................................................................................................................14
Inspecteren van de platen en het voorbereiden van de cel suspensie........................................14
Colony PCR & Agarose gel elektroforese.....................................................................................14
Inoculeren in het weekend..........................................................................................................15
Plasmid DNA isolation & measuring DNA concentration.............................................................15
DNA-digestie (BglII en SmaI) & Agarose gel elektroforese...........................................................15
Sanger sequencing & analyse......................................................................................................15
Logboek...........................................................................................................................................16
Voorbereiden van agar platen.....................................................................................................16
Inspecteren van platen en het voorbereiden van cel suspensie..................................................17
Colony PCR:..................................................................................................................................18
Agarose gel elektroforese:...........................................................................................................19
Inoculeren in het weekend..........................................................................................................19
Plasmide DNA-isolatie..................................................................................................................19
DNA-concentratie meten.............................................................................................................19
Sanger sequencing.......................................................................................................................20
DNA-digestie................................................................................................................................20
Agarose gel elektroforese............................................................................................................21
Resultaten........................................................................................................................................21
Inspecteren van platen................................................................................................................21
Agarose gel elektroforese – Na Colony PCR:................................................................................22
DNA-concentratie meten – Na plasmid DNA isolatie...................................................................22
Agarose gel elektroforese – Na DNA digestie:.............................................................................22
Sanger sequence..........................................................................................................................23
Conclusie/discussie..........................................................................................................................23
Transformatie..............................................................................................................................23
Colony PCR...................................................................................................................................23
DNA-concentratie meting – Na plasmid DNA-isolatie..................................................................24
DNA-digestie................................................................................................................................24
Sanger sequence..........................................................................................................................25
Verwerven en testen van eiwitten.......................................................................................................26
Titel..................................................................................................................................................26
Onderzoeksvraag.............................................................................................................................26
Hypothese........................................................................................................................................26
, Doel.................................................................................................................................................26
Voorspellingen.................................................................................................................................26
Het induceren van RnPP5-FL en RnPP5-dTPR eiwit expressie en lyseren van de E. coli cellen.. . .26
His-tag purificatie.........................................................................................................................26
Mass spectrometrie analyse........................................................................................................26
SDS-PAGE.....................................................................................................................................26
Fosfaat assay................................................................................................................................27
Materiaal en Methode.....................................................................................................................28
Her inoculeren van cellen voor eiwit expressie...........................................................................28
Het induceren van RnPP5 expressie en het lyseren van cellen....................................................28
His-tag purificatie.........................................................................................................................29
Mass spectrometry analyse.........................................................................................................29
SDS-PAGE.....................................................................................................................................29
Bradford eiwit assay....................................................................................................................30
Fosfatase assay............................................................................................................................30
Logboek...........................................................................................................................................30
Induceren van eiwit expressie en lyseren van cellen...................................................................30
SDS-PAGE.....................................................................................................................................31
His-tag purificatie.........................................................................................................................31
Bradford protein assay.................................................................................................................32
Coomassie staining......................................................................................................................34
Western blot................................................................................................................................34
Fosfatase assay............................................................................................................................34
Resultaten........................................................................................................................................36
MASCOT.......................................................................................................................................36
Coomassie staining......................................................................................................................37
Western blot................................................................................................................................37
Bradford protein assay.................................................................................................................38
Fosfatase assay............................................................................................................................39
Conclusie en discussie......................................................................................................................40
MASCOT.......................................................................................................................................40
Coomassie staining......................................................................................................................41
Western blot................................................................................................................................41
Fosfatase assay............................................................................................................................42
Overkoepelend onderzoek..................................................................................................................42
Titel..................................................................................................................................................42
Moleculaire techniek
Het effect van TPR op PP5
Een onderzoek naar de functie van het regulatoire tetratricopeptide repeat (TPR) domein op de
fosfoatase enzym activiteit van het eiwit fosfatase 5 (PP5) in Rattus norvegicus (RnPP5)
Femke te Dorsthorst
Studentennummer: 13120107
,Inhoud
Gene editing..........................................................................................................................................5
Titel....................................................................................................................................................5
Onderzoeksvraag...............................................................................................................................5
Hypothese..........................................................................................................................................5
Doel...................................................................................................................................................5
Voorspellingen...................................................................................................................................5
Agarose gel elektroforese..............................................................................................................5
DNA-purificatie..............................................................................................................................5
Golden Gate Assembly...................................................................................................................6
Materiaal en Methode.......................................................................................................................6
PCR.................................................................................................................................................6
Agarose gel elektroforese..............................................................................................................7
DNA-purificatie & DNA concentratie meting.................................................................................7
Golden Gate Assembly...................................................................................................................7
Logboek.............................................................................................................................................8
PCR:................................................................................................................................................8
Agarose gel elektroforese:.............................................................................................................8
Golden Gate Assembly:..................................................................................................................9
Resultaten........................................................................................................................................10
Elektroforese:..............................................................................................................................10
DNA-purificatie............................................................................................................................10
Conclusie/Discussie.........................................................................................................................10
Transformatie......................................................................................................................................11
Titel..................................................................................................................................................11
Onderzoeksvraag.............................................................................................................................11
Hypothese........................................................................................................................................11
Doel.................................................................................................................................................11
Voorspellingen.................................................................................................................................11
Transformeer E.coli......................................................................................................................11
Plate en incubate.........................................................................................................................11
Colony PCR & Agarose gel electrophorese...................................................................................12
Mini-prep plasmid DNA isolation.................................................................................................12
DNA digestie................................................................................................................................12
Sanger sequence..........................................................................................................................13
, Materiaal en Methode.....................................................................................................................13
Transformeer E. coli BL21............................................................................................................14
Plate en incuberen.......................................................................................................................14
Inspecteren van de platen en het voorbereiden van de cel suspensie........................................14
Colony PCR & Agarose gel elektroforese.....................................................................................14
Inoculeren in het weekend..........................................................................................................15
Plasmid DNA isolation & measuring DNA concentration.............................................................15
DNA-digestie (BglII en SmaI) & Agarose gel elektroforese...........................................................15
Sanger sequencing & analyse......................................................................................................15
Logboek...........................................................................................................................................16
Voorbereiden van agar platen.....................................................................................................16
Inspecteren van platen en het voorbereiden van cel suspensie..................................................17
Colony PCR:..................................................................................................................................18
Agarose gel elektroforese:...........................................................................................................19
Inoculeren in het weekend..........................................................................................................19
Plasmide DNA-isolatie..................................................................................................................19
DNA-concentratie meten.............................................................................................................19
Sanger sequencing.......................................................................................................................20
DNA-digestie................................................................................................................................20
Agarose gel elektroforese............................................................................................................21
Resultaten........................................................................................................................................21
Inspecteren van platen................................................................................................................21
Agarose gel elektroforese – Na Colony PCR:................................................................................22
DNA-concentratie meten – Na plasmid DNA isolatie...................................................................22
Agarose gel elektroforese – Na DNA digestie:.............................................................................22
Sanger sequence..........................................................................................................................23
Conclusie/discussie..........................................................................................................................23
Transformatie..............................................................................................................................23
Colony PCR...................................................................................................................................23
DNA-concentratie meting – Na plasmid DNA-isolatie..................................................................24
DNA-digestie................................................................................................................................24
Sanger sequence..........................................................................................................................25
Verwerven en testen van eiwitten.......................................................................................................26
Titel..................................................................................................................................................26
Onderzoeksvraag.............................................................................................................................26
Hypothese........................................................................................................................................26
, Doel.................................................................................................................................................26
Voorspellingen.................................................................................................................................26
Het induceren van RnPP5-FL en RnPP5-dTPR eiwit expressie en lyseren van de E. coli cellen.. . .26
His-tag purificatie.........................................................................................................................26
Mass spectrometrie analyse........................................................................................................26
SDS-PAGE.....................................................................................................................................26
Fosfaat assay................................................................................................................................27
Materiaal en Methode.....................................................................................................................28
Her inoculeren van cellen voor eiwit expressie...........................................................................28
Het induceren van RnPP5 expressie en het lyseren van cellen....................................................28
His-tag purificatie.........................................................................................................................29
Mass spectrometry analyse.........................................................................................................29
SDS-PAGE.....................................................................................................................................29
Bradford eiwit assay....................................................................................................................30
Fosfatase assay............................................................................................................................30
Logboek...........................................................................................................................................30
Induceren van eiwit expressie en lyseren van cellen...................................................................30
SDS-PAGE.....................................................................................................................................31
His-tag purificatie.........................................................................................................................31
Bradford protein assay.................................................................................................................32
Coomassie staining......................................................................................................................34
Western blot................................................................................................................................34
Fosfatase assay............................................................................................................................34
Resultaten........................................................................................................................................36
MASCOT.......................................................................................................................................36
Coomassie staining......................................................................................................................37
Western blot................................................................................................................................37
Bradford protein assay.................................................................................................................38
Fosfatase assay............................................................................................................................39
Conclusie en discussie......................................................................................................................40
MASCOT.......................................................................................................................................40
Coomassie staining......................................................................................................................41
Western blot................................................................................................................................41
Fosfatase assay............................................................................................................................42
Overkoepelend onderzoek..................................................................................................................42
Titel..................................................................................................................................................42
