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Post Type: Note
| Reagent | 1 | +ve ctrl |
| 5025/11 | 30 μL | - |
| p042 | - | 10 μL |
| H2O | - | 5 μL |
| NEB4 | 4 μL | 2 μL |
| BSA | 4 μL | 2 μL |
| EcoRI | 2 μL | 1 μL |
| NcoI | 2 μL | 1 μL |
Library 3
Round 1
Mutagenesis of plasmid p042 (4880/20)
Digestion of 4880/20 and the retrieved uncut plasmid from 4880/27 (4880/28)
Ligation of 4880/28.1 into digested pBAD/His (4880/29)
Transformation of plasmid 4880/29 into XL1 Blue (4880/30)
Electroporation 3 of 4880/30 into BW25141 (4880/35.1-7)
Round 2
(Round 2) mutagenesis on plasmid 4880/35 (4880/36)
Digestion of 4880/36 using EcoRI and NcoI (4880/37)
Ligation of 4880/37 into pBAD/His method 2 (Lyo-LigaseTM) (4880/39.2) and Ligation of 4880/37 into pBAD/His method 1 (4880/39.1)
Transformation of plasmids 4880/39.1 and 4880/39.2 into XL1 blue (4880/40)
Electroporation of 4880/40 into BW25141 (4880/41)
X-glu assay of electroporation 4880/41
Plasmid preparation from electroporations 4880/41.1-4
Round 3
Digestion of 4880/42 (4880/43)
Ligation of 4880/43 into pBAD/His (4880/44), Ligation of 4880/43 into pBAD/His (4880/49) and Test ligation conditions (4880/46)
Transformation of 4880/44 into XL1 Blue (4880/45), Transformation of 4880/46 into XL1 Blue (4880/47) and Transformation of 4880/49 into XL1 Blue (4880/50)
Electroporation: repeat of 4880/51 and 4880/52 (4880/53)
X-glu and X-gal assays for electroporation 4880/53
Round 4
R4 Mutagenesis 5025/11
Digestion 5025/14 - round 4
Ligation 5025/15 - round 4
Transformation 5025/16 (round 4) and Transformation 5025/17 - round 4
Electroporation 5025/18 (round 4)
Library 4
Round 1
Mutagenesis 5025/23 (experiment 2, round 1, attempt 2)
Digestion 5025/24 (experiment 2, round 1, attempt 2)
Ligation 5025/25 (experiment 2, round 1, attempt 2)
Transformation 5025/26 (experiment 2, round 1, attempt 2)
Electroporation 5025/27 (experiment 2, round 1)
Round 2
Mutagenesis 5025/28 (experiment 2, round 1)
Digestion 5025/29 (experiment 2, round 2)
Ligation 5025/32 (experiment 2, round 2, attempt 2)
Transformation 5025/33 (experiment 2, round 2, attempt 2)
Electroporation 5025/34 (experiment 2, round 2)
Round 3
Mutagenesis 5025/35 (experiment 2, round 3)
Digestion 5025/37 (experiment 2, round 3)
Ligation 5025/39 (experiment 2, round 3)
Transformation 5025/40 (experiment 2, round 3)
Electroporation 5025/41 (experiment 2, round 3)
Round 4
Mutagenesis 5025/42 (experiment 2, round 4)
Digestion 5025/43 (experiment 2, round 4)
Ligation 5025/44 (experiment 2, round 4)
Transformation 5025/45 (experiment 2, round 4)
Electroporation 5025/46 (experiment 2, round 4)
5025/52 Protein growth and purification of strains 5025/46.b1 (2nd batch) and 5025/46.b2 and 5025/53 Purification of protein 5025/46.b1
Sequencing 5025/48 of plasmids from 5025/46 (experiment 2, round 4) and 5025/48 Sequencing analysis - version 2
Activity assay 5025/56: activity of mutant 5025/46.b1 second assay and Activity assay 5025/63 measuring how the mutations have affected the β-glucuronidase activity of mutant 5025/46.b1
Electroporation and screen of native β-glucuronidase against alternative substrates to compare with 5025/66
Electroporation and alternative substrate screen 5025/66
Electroporation 5025/69 and alternative substrate screening of Wendy's round 4 and round 5 expts and my round 1
Round 1
Mutagenesis of plasmid p042 (4880/20)
Digestion of 4880/20 and the retrieved uncut plasmid from 4880/27 (4880/28)
Ligation of 4880/28.1 into digested pBAD/His (4880/29)
Transformation of plasmid 4880/29 into XL1 Blue (4880/30)
Electroporation 3 of 4880/30 into BW25141 (4880/35.1-7)
Round 2
(Round 2) mutagenesis on plasmid 4880/35 (4880/36)
Digestion of 4880/36 using EcoRI and NcoI (4880/37)
Ligation of 4880/37 into pBAD/His method 2 (Lyo-LigaseTM) (4880/39.2) and Ligation of 4880/37 into pBAD/His method 1 (4880/39.1)
Transformation of plasmids 4880/39.1 and 4880/39.2 into XL1 blue (4880/40)
Electroporation of 4880/40 into BW25141 (4880/41)
X-glu assay of electroporation 4880/41
Plasmid preparation from electroporations 4880/41.1-4
Round 3
Digestion of 4880/42 (4880/43)
Ligation of 4880/43 into pBAD/His (4880/44), Ligation of 4880/43 into pBAD/His (4880/49) and Test ligation conditions (4880/46)
Transformation of 4880/44 into XL1 Blue (4880/45), Transformation of 4880/46 into XL1 Blue (4880/47) and Transformation of 4880/49 into XL1 Blue (4880/50)
Electroporation: repeat of 4880/51 and 4880/52 (4880/53)
X-glu and X-gal assays for electroporation 4880/53
Round 4
R4 Mutagenesis 5025/11
Digestion 5025/14 - round 4
Ligation 5025/15 - round 4
Transformation 5025/16 (round 4) and Transformation 5025/17 - round 4
Electroporation 5025/18 (round 4)
Library 4
Round 1
Mutagenesis 5025/23 (experiment 2, round 1, attempt 2)
Digestion 5025/24 (experiment 2, round 1, attempt 2)
Ligation 5025/25 (experiment 2, round 1, attempt 2)
Transformation 5025/26 (experiment 2, round 1, attempt 2)
Electroporation 5025/27 (experiment 2, round 1)
Round 2
Mutagenesis 5025/28 (experiment 2, round 1)
Digestion 5025/29 (experiment 2, round 2)
Ligation 5025/32 (experiment 2, round 2, attempt 2)
Transformation 5025/33 (experiment 2, round 2, attempt 2)
Electroporation 5025/34 (experiment 2, round 2)
Round 3
Mutagenesis 5025/35 (experiment 2, round 3)
Digestion 5025/37 (experiment 2, round 3)
Ligation 5025/39 (experiment 2, round 3)
Transformation 5025/40 (experiment 2, round 3)
Electroporation 5025/41 (experiment 2, round 3)
Round 4
Mutagenesis 5025/42 (experiment 2, round 4)
Digestion 5025/43 (experiment 2, round 4)
Ligation 5025/44 (experiment 2, round 4)
Transformation 5025/45 (experiment 2, round 4)
Electroporation 5025/46 (experiment 2, round 4)
5025/52 Protein growth and purification of strains 5025/46.b1 (2nd batch) and 5025/46.b2 and 5025/53 Purification of protein 5025/46.b1
Sequencing 5025/48 of plasmids from 5025/46 (experiment 2, round 4) and 5025/48 Sequencing analysis - version 2
Activity assay 5025/56: activity of mutant 5025/46.b1 second assay and Activity assay 5025/63 measuring how the mutations have affected the β-glucuronidase activity of mutant 5025/46.b1
Electroporation and screen of native β-glucuronidase against alternative substrates to compare with 5025/66
Electroporation and alternative substrate screen 5025/66
Electroporation 5025/69 and alternative substrate screening of Wendy's round 4 and round 5 expts and my round 1
Post Type: Note
The graphs for the residual β-glucuronidase activity had initial rates calculated from the graph beyond the straight part of the curve.
The straight part of the curve has been re-drawn and initial rates re-calculated. There was an obvious anomaly from one of the results so this was omitted before a Hanes-Woolf plot was prepared and the new kinetic parameters calculated.
The new kinetic parameters were calculated as follows:
Vmax = 1/graph gradient = 1/9.16 x 10-6 = 1.092 x 10-7 M.s-1
KM=Vmax X Y intercept = 1.092 x 10-7 X 674 = 7.36 x 10-5 M (73.6 μM)
k = Vmax/[E] = 1.092 x 10-7/5 x 10-9 = 21.84 s-1
k/KM = 21.84/7.36 x 10-5 = 296 x 103 s-1.M-1
New graphs and calculations can be found in the attached Excel file in sheet 1.
The graphs for the residual β-glucuronidase activity had initial rates calculated from the graph beyond the straight part of the curve.
The straight part of the curve has been re-drawn and initial rates re-calculated. There was an obvious anomaly from one of the results so this was omitted before a Hanes-Woolf plot was prepared and the new kinetic parameters calculated.
The new kinetic parameters were calculated as follows:
Vmax = 1/graph gradient = 1/9.16 x 10-6 = 1.092 x 10-7 M.s-1
KM=Vmax X Y intercept = 1.092 x 10-7 X 674 = 7.36 x 10-5 M (73.6 μM)
k
k
New graphs and calculations can be found in the attached Excel file in sheet 1.
Post Type: Note
These are the most up-to-date files and results for the kinetics of the β-glucuronidase mutant 5025/46.b1 (G245A/K370R/W529L)
There are annotated text boxes on most pages explaining the contents of the page and the pages have been named, hopefully with some clarity. If there is a problem with opening Excel 2007 documents I can upload a Excel 2003 compatible file.
Here is the maths used to obtain the kinetic parameters:
From the Hanes-Woolf plot for residual β-glucuronidase activity
Graph gradient = 1/V max therefore 1/graph gradient = V max
1/7.31 x106 mol dm-3.s-1 = 1.37 x 10-7 mol dm-3.s-1
Y intercept = KM/V max therefore KM = Y intercept X V max
KM = 3.21 x 10-3 s-1 X 1.37 x 10-7 mol dm-3.s-1 = 0.00044 mol dm-3 (0.44 mM)
k cat = V max/[E] = 1.37 x 10-7 mol dm-3.s-1/5 x 10-9 mol dm-3 = 27.4 s-1
kcat/KM = 27.4 s-1/0.00044 mol dm-3 = 62272 s-1.mol dm-3
From the Hanes-Woolf plot for β-galactosidase activity
Graph gradient = 1/V max therefore 1/graph gradient = V max
1/1.10 x107 mol dm-3.s-1 = 9.03 x 10-8 mol dm-3.s-1
Y intercept = KM/V max therefore KM = Y intercept X V max
KM = 1.02 x 105 s-1 X 9.03 x 10-8 mol dm-3.s-1 = 0.00921 mol dm-3 (9.21 mM)
k cat = V max/[E] = 9.03 x 10-8 mol dm-3.s-1/1 x 10-5 mol dm-3 = 0.00903 s-1
kcat/KM = 0.00903 s-1/0.00921 mol dm-3 = 0.98 s-1.mol dm-3
In summary this gives
These are the most up-to-date files and results for the kinetics of the β-glucuronidase mutant 5025/46.b1 (G245A/K370R/W529L)
There are annotated text boxes on most pages explaining the contents of the page and the pages have been named, hopefully with some clarity. If there is a problem with opening Excel 2007 documents I can upload a Excel 2003 compatible file.
Here is the maths used to obtain the kinetic parameters:
From the Hanes-Woolf plot for residual β-glucuronidase activity
Graph gradient = 1/V max therefore 1/graph gradient = V max
1/7.31 x106 mol dm-3.s-1 = 1.37 x 10-7 mol dm-3.s-1
Y intercept = KM/V max therefore KM = Y intercept X V max
KM = 3.21 x 10-3 s-1 X 1.37 x 10-7 mol dm-3.s-1 = 0.00044 mol dm-3 (0.44 mM)
k cat = V max/[E] = 1.37 x 10-7 mol dm-3.s-1/5 x 10-9 mol dm-3 = 27.4 s-1
kcat/KM = 27.4 s-1/0.00044 mol dm-3 = 62272 s-1.mol dm-3
From the Hanes-Woolf plot for β-galactosidase activity
Graph gradient = 1/V max therefore 1/graph gradient = V max
1/1.10 x107 mol dm-3.s-1 = 9.03 x 10-8 mol dm-3.s-1
Y intercept = KM/V max therefore KM = Y intercept X V max
KM = 1.02 x 105 s-1 X 9.03 x 10-8 mol dm-3.s-1 = 0.00921 mol dm-3 (9.21 mM)
k cat = V max/[E] = 9.03 x 10-8 mol dm-3.s-1/1 x 10-5 mol dm-3 = 0.00903 s-1
kcat/KM = 0.00903 s-1/0.00921 mol dm-3 = 0.98 s-1.mol dm-3
In summary this gives
| β-glucuronidase | β-galactosidase | |
| Vmax | 1.37 x 10-7 M.s-1 | 9.03 x 10-8 M.s-1 |
| kcat | 27.4 s-1 | 0.00903 s-1 |
| KM | 0.00044 M | 0.00921 M |
| Kcat/KM | 62272 s-1.M-1 | 0.98 s-1.M-1 |
Post Type: Note
I have set up some 10 mL overnights for some of Wendy's stocks.
I have three concerns:
1. I can't find any list in the database of any cell stocks for experiment 2 so I've only done those for experiment 1.
2. The cell stock for rounds 3 and 5 are both missing and I couldn't find them searching in other boxes.
3. At some stage the cell stock for round 4 has been stored upside down and has probably melted and re-frozen. It was already quite liquid when I took off the lid (despite immediately placing deep in ice) and the stock was stuck to the lid. I cannot guarantee that the cell stock for the round 4 will grow.
I have set up some 10 mL overnights for some of Wendy's stocks.
I have three concerns:
1. I can't find any list in the database of any cell stocks for experiment 2 so I've only done those for experiment 1.
2. The cell stock for rounds 3 and 5 are both missing and I couldn't find them searching in other boxes.
3. At some stage the cell stock for round 4 has been stored upside down and has probably melted and re-frozen. It was already quite liquid when I took off the lid (despite immediately placing deep in ice) and the stock was stuck to the lid. I cannot guarantee that the cell stock for the round 4 will grow.