IGEM:IMPERIAL/2009/Assays Protocols/Shopping
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
Sigma Products for Cellulase Assay:
No. S-8625 = Sodium Acetate, Trihydrate
No. S-3504 = Sigmacell Solution (Sigmacell)
No. 16-10 = Glucose (HK)
PAH
Reagents:
Tris CH1 Buffer
L-Phenylalanine Solution
Catalase Enzyme Solution
DL-Dithiothreitol Solution
6-Methyltetrahydropterine Solution
Sigma Products for PAH Assay:
Trizma Base, Sigma Prod. No. T-1503
L-Phenylalanine, Sigma Prod. No. P-2126
Catalase, Sigma Stock No. C-100
DL-Dithiothreitol, Sigma Prod. No. D-0632
DL-6-Methyl-5,6,7,8-Tetrahydropterine Dihydrochloride, Sigma Prod. No. M-4758
Trichloroacetic Acid, 6.1 N Solution, Sigma Stock No.490-10
Nitric Acid, Aldrich Stock No. 25811-3
Sodium Nitrite, Sigma Prod. No. S-2252
1-Nitroso-2-Naphthol, Sigma Prod. No. N-3765
Sodium Hydroxide Solution, 1.0 N, Sigma Stock No. 930-65
L-Tyrosine Free Base, Sigma Prod. No. T-3754
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 I U.
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 (o, 4, 8, 12 minutes) by placing the tubes in boiling water for 2 min.
UDP in the cooled reaction mixtures was determined by adding 0.9 ml 40 mM-HEPES/ KOH (pH 6-8) containing 10 mM-MgCl,, 2.5 mM-phosphoenolpyruvate and 0.24 mM-NADH, centrifuging to remove precipitated protein, and measuring 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 lo%, and the mean was used. However, when a significant amount of substrate was consumed ( 2 15% at initial concentrations below K,,,), the rate from each time interval was handled separately and plotted against the average substrate concentration in the time interval, as recommended by Click 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:
OtsB Assay
Simple Assay for T6PS (OtsA) and T6PP (OtsB) [1]
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.
Shopping List
- Food grade sodium acetate : Sigma Purchasing Page
