Selected Cell
Cell:
Value:
Meta data
Organism
Assay Composition
Km Values
A
B
C
D
E
F
G
H
I
J
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
| Item | Value | ||||||||
| Experimentator | Wayne Aubrey | ||||||||
| Raw data | |||||||||
| Reference | SYSMO-LAB | ||||||||
| Description | Heterologous Expression of LDHs from L.lactis (MG1263) in Escherichia coli DH5α. Measurements on kinetics of L-LDHs at pH 7.5 | ||||||||
| Aim | Assessment of kinetic parameters of LDH to include in a catabolic model | ||||||||
| overexpression strain | E.coli DH5α | ||||||||
| Organism of Origin | L.lactis | ||||||||
| Vessel type | 96 wellplate | ||||||||
| Stirring rate | 0 | RPM | |||||||
| Temperature | 30 | degree celcius | |||||||
| Volume | 300 | µl | |||||||
| Gas flow | 0 | ml | |||||||
| pH | 7.5 | ||||||||
| SOP's used | 1 | ||||||||
| Explanations | Format | Data | |||||||
| TOPIC | |||||||||
| Title | text | Heterologous Expression of LDHs from L.lactis in Escherichia coli DH5α. Measurements on kinetics of L-LDHs at pH 7.5 | |||||||
| Comments | text | some comments | |||||||
| ASSAY | |||||||||
| Title | text | LDH kinetics | |||||||
| Assay_type | integer | enzyme kinetics | |||||||
| Comments | purified enzyme | ||||||||
| EXPERIMENT | |||||||||
| Experiment_description | text | Forward reaction: Measurement at 380 nm-> Decrease of NADH | |||||||
| Experiment_type | text | Concentration series | |||||||
| Experimentalist | text | Wayne Aubrey | |||||||
| Begin_date | date | 2012-11-19T0:0:0+0000 | |||||||
| User | integer | Wayne Aubrey | |||||||
| Group | integer | SYSMOLAB | |||||||
| SOP | 1 | ||||||||
| Organism of Origin | |||||||||
| Organism | integer | L.lactis | |||||||
| NCBI_ID | integer | 416870 | |||||||
| Strain | text | MG1363 | |||||||
| Genotype | text | wild-type | |||||||
| Phenotype | text | wild-type | |||||||
| Experimental_conditions | |||||||||
| FACTORS_STUDIED | |||||||||
| Item | text | Km | |||||||
| Start_value | float/text | 0 | |||||||
| End_value | float/text | 0 | |||||||
| Unit | text | mM | |||||||
| Comments | concentration dependent rates | ||||||||
| DATA_CREATED | |||||||||
| Datatype | text | rates | |||||||
| Creation_date | date | 11/2012-12/2012 | |||||||
| Status | text | row, normalised, .... | |||||||
| Person_created | text | Wayne Aubrey | |||||||
| DATA_FILE | |||||||||
| File_name | text | Llactis_ldh_kinetic | |||||||
| File_type | text | excel sheet | |||||||
| Storage_location | text | SYSMOSEEK | |||||||
| Storage_date | date | 2013-01-22T0:0:0+0000 | |||||||
| Last_change_date | date | 2013-01-22T0:0:0+0000 | |||||||
| TemplateID | text | Enzyme kinetics template |
A
B
C
D
E
F
G
H
I
J
K
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
| strain | gene | length | ORF | organism of origin | protein | EC number | MW in kDA | vector | tagged terminus | expression |
| E.coli DH5α IBA2_Lp_ldhL1 | ldhL1 | 978 bp | L0017 | L.lactis MG1363 | L-lactate dehydrogenase 1 | 1.1.1.27 | 34.21 | pASK-IBA2 | C | periplasmic |
A
B
C
D
E
F
G
H
I
J
K
L
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
| LDH | 1.1.1.27 | Cell extract dilutions | 10x-30x | Stock | M | Final conc in assay | M | mM | ||||
| LDH down | Master | HEPES | 0.3 | HEPES | 0.1 | M | 100 | |||||
| Reagent mix | 1 assay | 12 assay | 24 assay | 48 assay | Potassium | 1.314 | Potassium | 0.438 | M | 438 | ||
| (µL) | (µL) | (µL) | (µL) | glutamate | 1.2 | glutamate | 0.4 | M | 400 | |||
| H2Od | 124.8 | 1872 | 3949.4 | 6864 | phosphate | 0.003 | phosphate | 0.001 | M | 1 | ||
| Master Assay buffer | 100 | 1500 | 2800 | 5500 | NaCl | 0.15 | NaCl | 0.05 | M | 50 | ||
| CDM-PC Metals solution 1x | 30 | 450 | 840 | 1650 | Metals | 1 | x | Metals | 0.1 | x | ||
| MgSO4 | 1.2 | 18 | 33.6 | 66 | MgSO4 | 0.5 | MgSO4 | 0.002 | M | 2 | ||
| Fructose-1,6-bisphosphate | 3 | 45 | 84 | 165 | F16bP | 0.3 | F16bP | 0.003 | M | 3 | ||
| NADH (grade II disodium-) | 6 | 90 | 168 | 330 | NADH | 0.015 | NADH | 3.0E-4 | M | 0.3 | ||
| Sum | 265 | 3975 | 7875 | 14575 | PYR | 0.2 | PYR | 0.02 | M | 20 | ||
| Add per well | 265 | 265 | 265 | 265 | ||||||||
| Sample | 5 | 5 | 5 | 5 | PYR stock needed | |||||||
| Start reaction with: | 32 | 384 | 768 | 1536 | M | 0.2 | ||||||
| Pyruvate | 30 | 30 | 30 | 30 | mL | 1 | ||||||
| Total mixture in well | 300 | 300 | 300 | 300 | ||||||||
A
B
C
D
E
F
G
H
I
J
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
| Identifier | Sample | Organism | Enzyme | Subtrate | pH | Km value | standard deviation Km | Commentary | |
| unit | 1.0 | 1.0 | 1.0 | 1.0 | mM | % | |||
| 1 | L.lactis MG1363 | L-LDH | NADH | 7.5 | 0.125 | 0.006 | in presence of fructose-1.6-bisphosphate | ||
| 2 | L.lactis MG1363 | L-LDH | Pyruvate | 7.5 | 1.76 | 0.12 | in presence of fructose-1.6-bisphosphate | ||
