Team:Imperial College London/Wetlab/Protocols/Trehalose

=Trehalose Testing Assay=

Rationale
We want to monitor real time rate of production of trehalose per cell. To do this, we need two pieces of information. Firstly, we need to monitor cell growth to know the approximate cell count. This can be done by OD to obtain cell count. Secondly, we need to know the rate of production of trehalose. This can be obtained from performing the trehalose assay at regular time intervals of 30 minutes. From the second piece of information, we can plot a graph of trehalose concentration against time. The gradient of this graph would give us the total activity of cells. Dividing this by the number of cells will give us activity per cell per unit time. This should resemble a hill function.

We can also perform the above mentioned two steps for different concentration of HSL. This will also allow us to characterise our biobrick.

 Method  1) After activation of the CRP sensitive promoter, take samples at regular intervals.
 * Take 0.5ml sample from the culture and perform OD(600) to get cell count. Make sure OD is within linear portion of cell growth.


 * Take another 1ml sample and lyse using a sonicator (pulsed sonication, in an ice bath to reduce heating.)
 * Measure trehalose content using the trehlose method shown below:

Trehalose assay
 * Take samples at 30 minute time intervals, until a constant level of trehalose is reached.
 * Plot the increase in trehalose concentration against time. This will allow characterisation of the trehalose output with POPs input.
 * Functionality of the trehalose can be determined by the trehalose functionality assay.

KITS
Kits suitable for performing 100 assays are available from Megazyme. The kits contain the full assay method plus:

Bottle 1: Imidazole buffer (45 mL, 2 M, pH 7.0) plus magnesium chloride (100 mM) and sodium azide (0.02 % w/v) as a preservative. Stable for > 2 years at 4°C. Bottle 2: NADP+ (150 mg) plus ATP (440 mg). Stable for > 5 years at -20°C. Bottle 3: Hexokinase (425 U/mL) plus glucose-6-phosphate dehydrogenase (212 U/mL) suspension, 2.25 mL. Stable for > 2 years at 4°C. Bottle 4: Trehalase suspension (2.25 mL, 490 U/mL). Stable for > 2 years at 4°C. Bottle 5: D-Glucose standard solution (5 mL, 0.4 mg/mL) in 0.2 % (w/v) benzoic acid. Stable for > 4 years at room temperature. Bottle 6: Trehalose dihydrate control (~ 2 g). Stable for > 4 years at room temperature.

PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS
1. Use the contents of bottle 1 as supplied. Stable for > 2 years at 4°C. 2. Dissolve the contents of bottle 2 in 12 mL of distilled water. Divide into appropriately sized aliquots and store in polypropylene tubes at -20°C between use and cool during use. Once dissolved, the reagent is stable for > 2 years at -20°C. 3 & 4. Use the contents of bottles 3 and 4 as supplied. Before opening for the first time, shake the bottles to remove any protein that may have settled on the rubber stopper. Subsequently, store the bottles in an upright position. Stable for > 4 years at 4°C. 5. Use the contents of bottle 5 as supplied. Stable for > 4 years at room temperature. 6. Dissolve 200 mg of trehalose dihydrate (which corresponds to approx. 179 mg of trehalose) in 1 litre of distilled water. Add 0.2 mL of this solution to assays to check the activity of trehalase enzyme. Stable for > 4 years at -20°C.

NOTE: The D-glucose standard solution (solution 4) and the trehalose preparation (solution 5) are only assayed when there is some doubt about the accuracy of the spectrophotometer being used or where it is suspected that inhibition is being caused by substances in the sample. The concentration of trehalose is determined directly from the extinction coefficient of NADPH.

REQUIRED REAGENTS (not enclosed)
1. Reagent 1: Sodium hydroxide (50 mM) Dissolve 2.0 g of sodium hydroxide in 900 mL of distilled water. Adjust the volume to 1 litre. Store at room temperature. 2. Reagent 2: Alkaline borohydride (10 mg/mL sodium borohydride in 50 mM sodium hydroxide) Accurately weigh approx. 50 mg of sodium borohydride (Sigma Cat. No. S-9125) into polypropylene containers (10 mL volume with screw cap). Record the exact weight on the tubes, seal the tubes and store them in a desiccator with silica gel for future use. When weighing the borohydride, it is suggested that about 10 lots are prepared for convenience. Immediately before use, dissolve the sodium borohydride to a concentration of 10 mg/mL in 50 mM sodium hydroxide (Reagent 1). This solution is stable for 4-5 hours at room temperature. 3. Reagent 3: Acetic acid (200 mM) Add 11.6 mL of glacial acetic acid to 600 mL of distilled water and adjust the volume to 1 litre. Store at room temperature.

EQUIPMENT (RECOMMENDED)
1. Glass test tubes (round bottomed; 16 x 100 mm). 2. Disposable plastic cuvettes (1 cm light path, 3.0 mL). 3. Volumetric flasks (50 and 100 mL capacity). 4. Micro-pipettors, e.g. Gilson Pipetman® (20 μL and 100 μL). 5. Positive displacement pipettor e.g. Eppendorf Multipette® - with 5.0 mL Combitip® (to dispense 0.2 mL aliquots of imidazole buffer and 0.1 mL aliquots of NADP+/ATP solution). - with 25 mL Combitip® (to dispense 2.0 mL aliquots of distilled water). 6. Analytical balance. 7. Spectrophotometer set at 340 nm. 8. Vortex mixer (e.g. IKA®Yellowline Test Tube Shaker TTS2). 9. Whatman No.1 (9 cm) filter papers.

PREPARATION OF SAMPLE EXTRACTS and REMOVAL OF FREE D-GLUCOSE
A. Trehalose Extraction: Mill dry samples to pass a 0.5 mm screen. ie: strainer with square holes of sides 0.5mm Samples containing 0-12 % trehalose 1. Accurately weigh 1.0 g of the sample into a 100mL dry pyrex beaker and add 40 mL of 80°C hot distilled water. Place the beaker on a magnetic stirrer and stir and for 15 min. (i.e. until the sample is completely dispersed). 2. Quantitatively transfer the solution to a 50 mL volumetric flask. Adjust the volume to the mark with distilled water, mix the contents thoroughly and allow to cool to room temperature. Re-check the volume and adjust to the mark with distilled water if necessary.  Samples containing 12-40 % trehalose   1. Accurately weigh approx. 500 mg of the sample into a 100mL dry pyrex beaker and add 80 mL of hot distilled water at 80°C. Place the beaker on a magnetic stirrer and stir for 15 min (i.e. until the sample is completely dispersed. 2. Quantitatively transfer the solution to a 100 mL volumetric flask. Adjust the volume to the mark with distilled water, mix the contents thoroughly and allow to cool to room temperature. Re-check the volume and adjust to the mark with distilled water if necessary.  Further treatment of extracts  3. Filter an aliquot of the solution through a Whatman No. 1 (9 cm) filter circle. This solution may be slightly turbid, depending on the nature of the sample extracted. Analyse this solution immediately, or store at 4°C until analysed. If a turbidity forms in the solution, filter it again before analysis.  B. Removal of Reducing Sugars: a. Accurately dispense a 0.2 mL aliquot of the solution to be analysed (containing approx. 0.2 to 2.4 mg/mL of trehalose) into the bottom of a glass test-tube (16 x 100 mm). 8 Note: For samples containing 40-100 % trehalose, adjust the volume to 250 mL with DI water. b. Add 0.2 mL of Reagent 2 (alkaline borohydride solution) to the tube, stir the mixture vigorously and store at 40°C for 30 min to effect complete reduction of reducing-sugars to sugar alcohols. c. Add 0.5 mL of Reagent 3 (200 mM acetic acid) to the tube with vigorous stirring on a vortex mixer. A vigorous effervescence should be observed. (This treatment removes excess borohydride and adjusts the pH to approx. 4.5.) d. After 5 min, add 0.2 mL of solution 1 (2 M imidazole buffer, pH 7.0) to adjust the pH of the solution to approx. 7. This is the sample solution and should be analysed as described on page 5.

PROCEDURE
Wavelength: 340 nm Cuvette: 1 cm light path (glass or plastic) Temperature: ~ 25°C Final volume: 2.54 mL Sample solution: 4-80 μg of trehalose or D-glucose per cuvette (in 0.20 mL sample volume) Read against air (without a cuvette in the light path) or against water

1)

2) Mix*, read the absorbances at 340nm, of the solutions (A1) after 5 min and start the reactions by addition of:

3) Mix*, read the absorbances at 340nm, of the solutions (A2) at the end of the reaction (approx. 5 min). If the reaction has not stopped after 5 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min**.

(*) for example with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette cap or Parafilm®. (**) if the absorbance continues to increase, this may be due to effects of colour compounds or enzymes in the sample. These interfering substances may be removed during sample preparation.

SAMPLE PREPARATION
1. Sample dilution: The amount of trehalose present in the cuvette (i.e. in the 0.2 mL of sample being analysed) should range between 4 and 80 μg. Thus, the sample solution before the borohydride reduction step must be diluted sufficiently to yield a trehalose concentration between 0.12 and 2.4 g/L.

Dilution Table

If the value of DAtrehalose is too low (e.g. < 0.100), weigh out more sample or dilute less strongly.

NOTE: These calculations can be simplified by using the Megazyme Mega-CalcTM, downloadable from where the product appears in the Megazyme web site (www.megazyme.com).