LABJOURNAL
Charlotte van der Voort
13653318
The effect of TRP on PP5
A scientific investigation of the function of the
tetratricopeptide repeat (TPR) domain on the
phosphatase activity of the enzyme
phosphatase (PP5) in Rattus norvegicus
,Content
Gene editing…………………………………………………………………………………………………….4
Title…………………………………………………………………………………………………………………4
Goal…………………………………………………………………………………………………………………4
Predictions and/or expectations…………………………………………………………………………4
Agarose gel electroforesis………………………………………………………………………..4
Experimental design………………………………………………………………………………………….5
PCR ..……………………………………………………………………………………………….……5
Agarose gel electroforesis………………………………………………………………………..5
Log-content………………………………………………………………………………………………………6
PCR.………………………………………………………………………………………………….…..6
Agarose gel electroforesis………………………………………………………………………..7
Results……………………………………………………………………………………………………………..7
Agarose gel electroforesis ……………………………………………………………………….7
Conclusion/ discussion ……………………………………………………………………………………..8
Transformation…………………………………………………………………………………………………9
Title………………………………………………………………………………………………………………….9
Goal………………………………………………………………………………………………………………….9
Predictions and/or expectations………………………………………………………………………….9
DNA purification…………………………………………………………………………………….9
Golden Gate assembly.…………………………………………………………………………….9
Transform E.coli BL21 Gold.…………………………………………………………………..10
Agarose gel electroforesis after colony PCR....…………………………………………..10
Agarose gel electroforesis after DNA digestion..………………………………………..11
Sanger sequence………..…………………………………………………………………………..11
Experimental design………………………………………………………………………………………….11
DNA purification……………………………………………………………………………………12
Golden Gate assembly.……………………………………………………………………………12
Transform E.coli BL21 Gold.……………………………………………………………………12
Colony PCR……………………………………………………………………………………………13
Agarose gel electroforesis……………………......…………………………………..............13
Plasmid DNA isolatation…………………………………………………………………………13
Sanger sequence………..…………………………………………………………………………..14
Log-content………………………………………………………………………………………………………14
DNA purification……………………………………………………………………………………14
Measure DNA concentration after DNA purification…………………….……………14
Golden Gate assembly.……………………………………………………………………………15
Prepare agar plates…………………………………………………………………….…………..15
Transform E.coli BL21 Gold.………………………………………………………...............16
Colony PCR…………………………………………………………………………………………...16
Inspect plates and prepare cell suspension……………………………………………....18
Agarose gel electroforesis after colony PCR..……………………………………………..19
Reinoculate cells for protein expression……………………………………………………19
Plasmid DNA isolatation…………………………………………………………………………19
DNA digestion BglII/SmaI……………………………………………………………………..20
Measure DNA concentration after plasmid DNA isolation………………………….21
Agarose gel electroforesis after DNA digestion……………………………………….…21
Sanger sequence………..…………………………………………………………………………..21
2
RAPPORTTITEL
, Results………………………………………………………………………………………………………….…22
Agarose gel electroforesis after colony PCR.……………………………………………..22
Agarose gel electroforesis after DNA digestion………………………………………….23
Sanger sequence…………………………………………………………………………………….23
Conclusion/ discussion …………………………………………………………………………………….24
Protein expression……………………………………………………………………………………………26
Title………………………………………………………………………………………………………………..26
Goal………………………………………………………………………………………………………………..26
Predictions and/or expectations………………………………………………………………………..26
Induce protein expression and lyse cell…………………………………………............26
His-tag purification………………………………………………………………………………..26
Transform E.coli BL21 Gold.…………………………………………………………………...26
Mass spectrometry analysis....…………………………………………………….………..…26
SDS-PAGE ..…………………………………………………………………..……………………..26
Bradford assay ………..………………………………………………………….…….……….….26
Western blot ………..………………………………………………………………..……………...26
Coomassie staining………..…………..…………………………………………………………..27
Experimental design………………………………………………………………………………………...27
Induce protein expression and lyse cell………………….………………………………..28
SDS-PAGE ..………………………………………………………………………………………....28
Bradford assay ………..…………………………………………………………………………….28
Mass spectrometry analysis....……………………………………….………………………..29
Western blot ………..……………………………………………………………………………….29
Log-content……………………………………………………………………………………………………..29
Induce protein expression and lyse cell…………………………………….……………...29
SDS-PAGE ..………………………………………………………………………………….……...30
Prepare for Mass spectrometry…………………………………………………………………31
His-tag purification…………………………………………………………………………………31
Bradford assay ………..……………………………………………………………………………..31
SDS-PAGE ..…………………………………………………………………………………………..34
Western blot ………..………………………………………………………………………………..34
Coomassie staining………..………..……………………………………………………………..34
Mass spectrometry analysis....…………………………………….…………………………..35
Results…………………………………………………………………………………………………………….35
Coomassie staining………..……………………..…………………………………………….….35
Western blot…………………………………………………………..……………………………..36
Mass spectrometry………………………………………………………………………………...36
Conclusion/ discussion …………………………………………………………………………………..…37
Phosphatase activity…………………………………………………………………………………………38
Title………………………………………………………………………………………………………………..38
Goal………………………………………………………………………………………………………………..38
Predictions and/or expectations………………………………………………………………………..38
Phosphatase assay………………………………………………………………………………...38
Experimental design…………………………………………………………………………………………38
Phosphatase assay…………………………………………………………………………………38
Log-content……………………………………………………………………………………………………..39
Phosphatase assay…………………………………………………………………………………39
Results…………………………………………………………………………………………………………….40
Phosphatase assay………………………………………………………………………………….40
Conclusion/ discussion …………………………………………………………………………………….41
3
RAPPORTTITEL
, Title
Developing an pET28-a vector with RnPP5-FL and RnPP5-ΔTPR, using a PCR and
Agarose gel electroforesis
Goal
Amplifying the DNA with the RnPP5-FL and RnPP5-ΔTPR DNA fragments and
ligating them into a pET28-a vector, using a PCR and Agarose gel electrophoresis, to
be used in the next experiment for the transformation of an E.coli BL12 Gold.
Predictions and/or expectations
- MPP PP5 C by phosphate domain(light green)
- PLN03088 by TPR domain (light blue)
- 4JA9 A (light pink)
- Active site (yellow)
- Metal binding site (magenta)
- TPR interaction site (red)
- Putative protein binding surface (orange)
Figure 1: The domains of the PP5
protein. This figure is made with
snapgene.
Agarose gel electroforesis
- The expected length of the PP5-FL PCR product is 1528 bp, while the expected
length of the PP5-ΔTPR PCR product should be 1051 bp.
- The primers in the blank will bind together and leave a vague band on the gel
as a result.
Figure 2: The expected length of the PP5-FL PCR product is 1528 bp. This figure is made with snapgene.
Figure 3: The expected length of the PP5-ΔTPR PCR product is 1051 bp. This figure is made with snapgene.
4
RAPPORTTITEL
Charlotte van der Voort
13653318
The effect of TRP on PP5
A scientific investigation of the function of the
tetratricopeptide repeat (TPR) domain on the
phosphatase activity of the enzyme
phosphatase (PP5) in Rattus norvegicus
,Content
Gene editing…………………………………………………………………………………………………….4
Title…………………………………………………………………………………………………………………4
Goal…………………………………………………………………………………………………………………4
Predictions and/or expectations…………………………………………………………………………4
Agarose gel electroforesis………………………………………………………………………..4
Experimental design………………………………………………………………………………………….5
PCR ..……………………………………………………………………………………………….……5
Agarose gel electroforesis………………………………………………………………………..5
Log-content………………………………………………………………………………………………………6
PCR.………………………………………………………………………………………………….…..6
Agarose gel electroforesis………………………………………………………………………..7
Results……………………………………………………………………………………………………………..7
Agarose gel electroforesis ……………………………………………………………………….7
Conclusion/ discussion ……………………………………………………………………………………..8
Transformation…………………………………………………………………………………………………9
Title………………………………………………………………………………………………………………….9
Goal………………………………………………………………………………………………………………….9
Predictions and/or expectations………………………………………………………………………….9
DNA purification…………………………………………………………………………………….9
Golden Gate assembly.…………………………………………………………………………….9
Transform E.coli BL21 Gold.…………………………………………………………………..10
Agarose gel electroforesis after colony PCR....…………………………………………..10
Agarose gel electroforesis after DNA digestion..………………………………………..11
Sanger sequence………..…………………………………………………………………………..11
Experimental design………………………………………………………………………………………….11
DNA purification……………………………………………………………………………………12
Golden Gate assembly.……………………………………………………………………………12
Transform E.coli BL21 Gold.……………………………………………………………………12
Colony PCR……………………………………………………………………………………………13
Agarose gel electroforesis……………………......…………………………………..............13
Plasmid DNA isolatation…………………………………………………………………………13
Sanger sequence………..…………………………………………………………………………..14
Log-content………………………………………………………………………………………………………14
DNA purification……………………………………………………………………………………14
Measure DNA concentration after DNA purification…………………….……………14
Golden Gate assembly.……………………………………………………………………………15
Prepare agar plates…………………………………………………………………….…………..15
Transform E.coli BL21 Gold.………………………………………………………...............16
Colony PCR…………………………………………………………………………………………...16
Inspect plates and prepare cell suspension……………………………………………....18
Agarose gel electroforesis after colony PCR..……………………………………………..19
Reinoculate cells for protein expression……………………………………………………19
Plasmid DNA isolatation…………………………………………………………………………19
DNA digestion BglII/SmaI……………………………………………………………………..20
Measure DNA concentration after plasmid DNA isolation………………………….21
Agarose gel electroforesis after DNA digestion……………………………………….…21
Sanger sequence………..…………………………………………………………………………..21
2
RAPPORTTITEL
, Results………………………………………………………………………………………………………….…22
Agarose gel electroforesis after colony PCR.……………………………………………..22
Agarose gel electroforesis after DNA digestion………………………………………….23
Sanger sequence…………………………………………………………………………………….23
Conclusion/ discussion …………………………………………………………………………………….24
Protein expression……………………………………………………………………………………………26
Title………………………………………………………………………………………………………………..26
Goal………………………………………………………………………………………………………………..26
Predictions and/or expectations………………………………………………………………………..26
Induce protein expression and lyse cell…………………………………………............26
His-tag purification………………………………………………………………………………..26
Transform E.coli BL21 Gold.…………………………………………………………………...26
Mass spectrometry analysis....…………………………………………………….………..…26
SDS-PAGE ..…………………………………………………………………..……………………..26
Bradford assay ………..………………………………………………………….…….……….….26
Western blot ………..………………………………………………………………..……………...26
Coomassie staining………..…………..…………………………………………………………..27
Experimental design………………………………………………………………………………………...27
Induce protein expression and lyse cell………………….………………………………..28
SDS-PAGE ..………………………………………………………………………………………....28
Bradford assay ………..…………………………………………………………………………….28
Mass spectrometry analysis....……………………………………….………………………..29
Western blot ………..……………………………………………………………………………….29
Log-content……………………………………………………………………………………………………..29
Induce protein expression and lyse cell…………………………………….……………...29
SDS-PAGE ..………………………………………………………………………………….……...30
Prepare for Mass spectrometry…………………………………………………………………31
His-tag purification…………………………………………………………………………………31
Bradford assay ………..……………………………………………………………………………..31
SDS-PAGE ..…………………………………………………………………………………………..34
Western blot ………..………………………………………………………………………………..34
Coomassie staining………..………..……………………………………………………………..34
Mass spectrometry analysis....…………………………………….…………………………..35
Results…………………………………………………………………………………………………………….35
Coomassie staining………..……………………..…………………………………………….….35
Western blot…………………………………………………………..……………………………..36
Mass spectrometry………………………………………………………………………………...36
Conclusion/ discussion …………………………………………………………………………………..…37
Phosphatase activity…………………………………………………………………………………………38
Title………………………………………………………………………………………………………………..38
Goal………………………………………………………………………………………………………………..38
Predictions and/or expectations………………………………………………………………………..38
Phosphatase assay………………………………………………………………………………...38
Experimental design…………………………………………………………………………………………38
Phosphatase assay…………………………………………………………………………………38
Log-content……………………………………………………………………………………………………..39
Phosphatase assay…………………………………………………………………………………39
Results…………………………………………………………………………………………………………….40
Phosphatase assay………………………………………………………………………………….40
Conclusion/ discussion …………………………………………………………………………………….41
3
RAPPORTTITEL
, Title
Developing an pET28-a vector with RnPP5-FL and RnPP5-ΔTPR, using a PCR and
Agarose gel electroforesis
Goal
Amplifying the DNA with the RnPP5-FL and RnPP5-ΔTPR DNA fragments and
ligating them into a pET28-a vector, using a PCR and Agarose gel electrophoresis, to
be used in the next experiment for the transformation of an E.coli BL12 Gold.
Predictions and/or expectations
- MPP PP5 C by phosphate domain(light green)
- PLN03088 by TPR domain (light blue)
- 4JA9 A (light pink)
- Active site (yellow)
- Metal binding site (magenta)
- TPR interaction site (red)
- Putative protein binding surface (orange)
Figure 1: The domains of the PP5
protein. This figure is made with
snapgene.
Agarose gel electroforesis
- The expected length of the PP5-FL PCR product is 1528 bp, while the expected
length of the PP5-ΔTPR PCR product should be 1051 bp.
- The primers in the blank will bind together and leave a vague band on the gel
as a result.
Figure 2: The expected length of the PP5-FL PCR product is 1528 bp. This figure is made with snapgene.
Figure 3: The expected length of the PP5-ΔTPR PCR product is 1051 bp. This figure is made with snapgene.
4
RAPPORTTITEL
