IGEM:IMPERIAL/2009/Assays Protocols/Shopping: Difference between revisions
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Enzymes to be assayed at 30°C. The amount of enzyme producing 1 umol product per minute in the respective standard assay is | Enzymes to be assayed at 30°C. The amount of enzyme producing 1 umol product per minute in the respective standard assay is IU. | ||
Samples were diluted, when necessary, with 50mMHEPES/ | Samples were diluted, when necessary, with 50mMHEPES/ | ||
| Line 53: | Line 53: | ||
* 1mg/ml bovine albumin. | * 1mg/ml bovine albumin. | ||
2) Start reactions with enzyme and stopped after set times ( | 2) Start reactions with enzyme and stopped after set times (0, 4, 8, 12 minutes) by placing the tubes in boiling water for 2 min. | ||
3) Add 0.9 ml 40 mM-HEPES/ KOH (pH 6-8) containing 10 mM-MgCl2, 2.5 mM-phosphoenolpyruvate | |||
and 0.24 mM-NADH | |||
KOH (pH 6-8) containing 10 mM- | |||
and 0.24 mM-NADH | 4) Centrifuge to remove precipitated protein, | ||
NADH on addition of pyruvate kinase and lactate dehydrogenase. | 5) Measure spectrophotometrically at 340 nm the disappearance of NADH on addition of pyruvate kinase and lactate dehydrogenase. | ||
When substrate concentrations were varied in TPS assays, rates at each | |||
concentration were measured over two different time intervals. They | When substrate concentrations were varied in TPS assays, rates at each concentration were measured over two different time intervals. They generally agreed within l0%, and the mean was used. However, when a significant amount of substrate was consumed ( >15% at initial concentrations below Km), the rate from each time interval was handled | ||
generally agreed within | separately and plotted against the average substrate concentration in the time interval, as recommended by Glick et al. (1979). | ||
significant amount of substrate was consumed ( | |||
concentrations below | |||
separately and plotted against the average substrate concentration in | |||
the time interval, as recommended by | |||
| Line 84: | Line 80: | ||
Reagents Required: <br> | Reagents Required: <br> | ||
===Trehalose Assay=== | ===Trehalose Assay=== | ||
| Line 114: | Line 109: | ||
<b> Membrane Staining Assay </b> <br> | <b> Membrane Staining Assay </b> <br> | ||
[http://openwetware.org/images/b/b9/Cell_Death_Assay.pdf Cell Membrane Staining Assay]: This assay uses a kit with certain stains to determine whether cells are alive or dead. The assay stains those cells with intact cell membranes green (the 'alive' cells), and those with damaged cell membranes red (the 'dead' cells). This is a quantitative measure of cell death. However, as we are using restriction enzymes, we will not directly affect the cell membrane, so this assay may not work. Natural degredation of the membrane without maintenance from the cell may occur, in which case this would be a useful guide. In either case, it would be useful as an indicator of the viability of the cells with disrupted genetic material. Another issue is the fact that our cells are to be encapsulated, so whether this staining technique can work through the colanic acid capsule is unclear. | [http://openwetware.org/images/b/b9/Cell_Death_Assay.pdf Cell Membrane Staining Assay]: This assay uses a kit with certain stains to determine whether cells are alive or dead. The assay stains those cells with intact cell membranes green (the 'alive' cells), and those with damaged cell membranes red (the 'dead' cells). This is a quantitative measure of cell death. However, as we are using restriction enzymes, we will not directly affect the cell membrane, so this assay may not work. Natural degredation of the membrane without maintenance from the cell may occur, in which case this would be a useful guide. In either case, it would be useful as an indicator of the viability of the cells with disrupted genetic material. Another issue is the fact that our cells are to be encapsulated, so whether this staining technique can work through the colanic acid capsule is unclear. | ||
Latest revision as of 04:42, 7 August 2009
Module 1
Cellulase
Reagents:
Sodium Acetate Buffer, pH 5
- Prepared using Sodium Acetate, Trihydrate, Sigma Prod. No. S-8625
Sigmacell Solution
Cellulase Enzyme Solution
Glucose Determination Vial
PAH
Reagents:
Tris CH1 Buffer
L-Phenylalanine Solution
Catalase Enzyme Solution
DL-Dithiothreitol Solution
6-Methyltetrahydropterine Solution
Module 2
Colanic Acid
Link to Assay Page
Protocol for Quantitative Assay
Qualitative Assay using electron microscopy could be performed as well, to ensure sufficient encapsulation.
Reagents:
No special reagents needed. Centrifuge performed on bacterial media.
Equipment:
PCV Centrifuge Tubes
OtsA
Simple Assay for T6PS (OtsA) [1]
Enzymes to be assayed at 30°C. The amount of enzyme producing 1 umol product per minute in the respective standard assay is IU.
Samples were diluted, when necessary, with 50mMHEPES/ KOH (pH7.0)/0-1 ~M-EDTA/mS g bovine albumin ml-I, because the TPS activity was unstable at low protein concentrations.
1) Make up standard assay mixtures (100 ul final volume) containing:
- 40 mM HEPES/ KOH (pH 6.8)
- 10 mM MgCl2
- 10 mM Glucose-6-Phosphate
- 5 mM UDPG
- 1mg/ml bovine albumin.
2) Start reactions with enzyme and stopped after set times (0, 4, 8, 12 minutes) by placing the tubes in boiling water for 2 min.
3) Add 0.9 ml 40 mM-HEPES/ KOH (pH 6-8) containing 10 mM-MgCl2, 2.5 mM-phosphoenolpyruvate and 0.24 mM-NADH
4) Centrifuge to remove precipitated protein,
5) Measure spectrophotometrically at 340 nm the disappearance of NADH on addition of pyruvate kinase and lactate dehydrogenase.
When substrate concentrations were varied in TPS assays, rates at each concentration were measured over two different time intervals. They generally agreed within l0%, and the mean was used. However, when a significant amount of substrate was consumed ( >15% at initial concentrations below Km), the rate from each time interval was handled separately and plotted against the average substrate concentration in the time interval, as recommended by Glick et al. (1979).
Trehalose 6-Phosphate Synthases Catalyses the following reaction:
GDP-glucose + glucose 6-phosphate --> GDP + alpha,alpha-trehalose 6-phosphate
Trehalose 6-Phosphate Phosphatase catalyses the following reaction:
alpha,alpha-trehalose 6-phosphate + H2O --> alpha,alpha-trehalose + phosphate
We will perform the quantitative assay shown above to assess the activity of T6PS and T6PP
Reagents Required:
Trehalose Assay
Trehalose Assay Method
Link to Assays Purchasing Page
We can test the final OtsAB construct using this simple trehalose assay. The assay is purchased as a kit, with simple step by step instructions to quantify the activity of the enzyme.
Module 3
Heat Induction
Link to Heat Induction Protocols
Visualising SDS -PAGE
Reagents:
Normal SDS-PAGE materials
Nitrocellulose Transfer Membrane
Coomassie Blue R-250
Dimethyl Sulphoxide
Killing Strategy
Measuring Cell Death by counting Colony Forming Units (CFUs)
CFU Method: By knowing the density of cells present in a media using OD measurements, and depositing a set amount of this media (and therefore a set no. of cells) onto an agar plate, we can know the proportion of viable cells still present in our colony after restriction enzyme production. This gives a quantifiable method for the calculation of the efficiency of our killing strategy.
Membrane Staining Assay
Cell Membrane Staining Assay: This assay uses a kit with certain stains to determine whether cells are alive or dead. The assay stains those cells with intact cell membranes green (the 'alive' cells), and those with damaged cell membranes red (the 'dead' cells). This is a quantitative measure of cell death. However, as we are using restriction enzymes, we will not directly affect the cell membrane, so this assay may not work. Natural degredation of the membrane without maintenance from the cell may occur, in which case this would be a useful guide. In either case, it would be useful as an indicator of the viability of the cells with disrupted genetic material. Another issue is the fact that our cells are to be encapsulated, so whether this staining technique can work through the colanic acid capsule is unclear.
