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 [1]
Trehalose 6-Phosphate Synthases Catalyses the following reaction:
GDP-glucose + glucose 6-phosphate --> GDP + alpha,alpha-trehalose 6-phosphate
We will perform the quantitative assay shown above to assess the activity of T6PS
Reagents Required:
Extraction Buffer containing;
Hepes, DTT, KCl, Cocktail inhibitor from Boerhinger
1g Glass beads to break up cells
Hepes, KcCl, EDTA, MgSO4, UDP Glucose
Glucose-6-phosphate
Lactate Dehydrogenase
Pyruvate Kinase
OtsB (Trehalose) Assay
Link to Assays Purchasing Page
Trehalose 6-Phosphate Phosphatase catalyses the following reaction:
alpha,alpha-trehalose 6-phosphate + H2O --> alpha,alpha-trehalose + phosphate
We will therefore perform a trehalose assay on the products of OtsB to see if functional T6PP is produced. This will give us a quantifiable output to the POPs input to the gene.
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
