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Team:HUNGENIOUS/Methods
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Revision as of 10:48, 12 June 2014
Methods
Chitinase assay
We ordered a chitinase assay kit from Sigma-Aldrich.
This chitinase assay kit is based on the enzymatic hydrolysis of chitinase substrates. It can be carried out at pH 4.8 and 37°C, so it is appropriate for our purposes, because our chitinase enzyme also has its highest relative efficiency rate at these properites. This hydrolysis releases p-nitrophenol (4-nitrophenol) [Picture1], which upon ionization in basic pH, can be measured colorimetrically at 405 nm.
Procedure:
1. Equilibrate the Substrate Solution and the Standard Solution to 37 °C by incubating for several minutes in a 37 °C water bath.
2. Set the plate reader at 405 nm.
3. Add the reaction components to the 96 well plate according to Table 1 and mix using a horizontal shaker or by pipetting. The substrate should be added first and the enzyme should be added last.
4. Incubate the plate for 30 minutes at 37 °C. If required, the incubation time for highly active samples can be reduced to as low as 5 minutes.
5. Stop the reactions by adding 200 ml of Stop Solution to each well, except for the wells containing the Standard Solution. After the addition of the Stop Solution the reaction mixture will develop a yellow tint.
6. Measure the absorption at 405 nm no later than 30 minutes after ending the reaction.
Gel electrophoresis
DNA is negatively charged due to the phosphate ions (PO43-) backbone. When an electric field is applied across an agarose matrix containing DNA, the nucleic acid fragments move towards the positive cathode. This migration of DNA is dependent upon the size of the matrix pores and the length of the DNA in question. For a fixed pore size and potential difference, a particular DNA fragment migrates a distance proportional to the molecular weight of the molecule. This allows DNA fragments to be separated by size. The sizes are calculated by comparison with a 'ladder' of standard DNA fragments of known sizes. We used the Cleaver Scientific Multi Sub Mini + 7x10 cm geltray.
Procedure:
1. For 1% agarose gel , add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10x TAE stock buffer with water).
2. Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
3. Ensure electrophoresis chamber is clean and dry. Slot in the comb.
4. Add the gel to the electrophoresis chamber, and wait to set. The comb can then be removed from the chamber.
5. Fill the electrophoresis apparatus half-full with 1x TAE buffer solution (for good electrical contact) and place the set gel in the buffer. Ensure that there are no air bubbles (particularly in the wells created by the comb).
6. Add the ladder solution to the first well, and the DNA samples to subsequent wells. A loading dye (DNA Electrophoresis Sample Loading Dye Bio-Rad #166-0401EDU) should be added to the mixtures to aid visualization when loading into wells.
7. Connect the electrodes to the apparatus (the right way!). Set DC voltage at 80V (with current at approximately 3 mA) and run for 30-60 minutes.
8. After the run, take the gel out and put it into a dyeing dish. Pure Fast Blast DNA Stain (Fast Blast DNA Stain #166-0420EDU) onto the top of the gel. Wait for 15-20 minutes.
9. Pour the dye off the gel, the separated DNA should be visible now.
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a method of resolving proteins of different molecular weights by mixing samples with SDS, loading samples into usually an acrylamide gel and passing an electric current through it.
SDS is an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass, allowing fractionation of proteins via electrophoresis similar to a DNA gel with longer proteins experiencing more difficulty moving through the gel than shorter proteins. Samples are often heated in boiling water prior to loading to shake up the molecules and allow improved binding with SDS. A dye, bromophenol blue is used to indicate the stopping point.
After electrophoresis the gel is rinsed in D.I water and stained with a dye, Coomassie Brilliant Blue for improving fainter bands for visualisation of the separated proteins. After staining the gel is rinsed again and left to de-stain to your desired amount either in D.I water or in a de-staining solution.
We ran our gels using Bio-Rad electrophoresis tanks.
Ingredients:
-100 µg protein per lane
-43.8 kDa (mass of the protein)
-15% slab gels
-samples:
-0.0625 M Tris-HCl (pH 6.8)
-2% (w/v) SDS
-5% (v/v) ß-mercaptoethanol
-10% (v/v) glycerol
-0.001% (w/v) bromophenol blue
-stained: 0.2% (w/v) Coomassie Brilliant Blue R250 in 50% methanol/10% acetic acid for 16 h
(or Simply Blue Safe Stain (Invitrogen))
-destaining: 12.5% isopropanol/10% acetic acid
Screening Chitinase-activity
We found this method in the article of N. Murthy, B. Bleakley: Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase- Producing Microorganisms.
We would like to use this method as a chitinase assay. In order to check that the chitinase gene was infiltrated and expressed in the genetically modified bacteria, we inoculate the bacteria onto colloidal chitin agar. ,,The colloidal chitin agar (pH 7.0+/-0.2) prepared had the following ingredients (in g/L): (K2HPO4, 0.7; KH2PO4, 0.3; MgSO4 X 5H2O, 0.5; FeSO4 X 7H20, 0.01; ZnSO4, 0.001; MnCl2, 0.001), amended with 2.0 % moist colloidal chitin. Bacto Agar (Difco) was added at 2.0% as a solidifying agent. The medium was sterilized by autoclaving at STP (standard temperature and pressure: 15 psi, 20 minutes, 121 ° C) and poured into sterile Petri plates (approximately 20 ml per plate).”
If the chitinase gene expresses, we’ll see clearing zones around the colonies on the colloidal chitin agar, indicating the production of chitinase. From the ratio of the clearing zone to colony diameter, we can conclude the efficiency of the gene expression.
