Team:Johns Hopkins/Notebook/VitProtocol

Vitamin C Concentration Assay

(By Anne Marie Helmenstine, Ph.D)

1. Add 25.00 ml of vitamin C standard solution to a 125 ml Erlenmeyer flask.
2. Add 10 drops of 1% starch solution.
3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.
4. Titrate the solution until the endpoint is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.
5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.
6. Repeat the titration at least twice more. The results should agree within 0.1 ml.
7. You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the endpoint.

Dough Media Recipes

Optimized recipe per plate:

• 20ml H₂O
• 0.24g NaCl
• 0.15g bactoagar
• 0.55g flour

High Performance Liquid Chromatography (HPLC)

We grew up yeasts in liquid culture, and took aliquots at different time points to determine the cell count and amount of beta carotene produced. The cell count was determined by measuring the optical density of the samples at 600nm. A calibration curve was used to estimate the number of cells by counting the number of cells counted on a hemocytometer was plotting it against their absorbance. Cell counts at different time points are then compared to it.

β-carotene was extracted from wild type and genetically engineered beta carotene producing yeasts for quantification. After addition of pyrogallol in methanol, cells were saponified with potassium hydroxide. The samples were incubated at 75 degrees for an hour. Beta carotene was then extracted by adding hexane to the samples and shaking them vigorously. Aliquots were then taken for spectroscopy and for drying. Concentration of beta carotene in hexane was determined from absorbance at 449nm on a UV spectrophotometer. Extracted products were verified and quantified by performing High Pressure Liquid Chromatography analysis on a C18 Varian 5 micron 5x250mm with a UV detector.

Extracts were dried in a vacuum desiccator and then dissolved in THF. Beta carotene was eluted isocratically with a mobile phase of acetonitrile:methanol:propanol (50:40:10) at 1ml/min and 450nm. Beta carotene has a retention time of 18 minutes. Extracts were compared to standards obtained from Fluka.

Vitamin C Assay Solutions

Starch Indicator Solution

1. Add 0.50 g soluble starch to 50 ml near-boiling distilled water.
2. Mix well and allow to cool before use. (doesn't have to be 1%; 0.5% is fine)

Iodine Solution

1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in 200 ml of distilled water.
2. Add 30 ml of 3 M sulfuric acid.
3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml with distilled water.
4. Mix the solution.
5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.

Vitamin C Standard Solution

1. Dissolve 0.250 g vitamin C (ascorbic acid) in 100 ml distilled water.
2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as your vitamin C standard solution.

Spectroscopy

Carotenoid Assay

• Suspend cells in 1 mL of sterile water.
• Add 0.5 to 0.75 mm glass beads and vortex for 3 minutes.
• Add 2.5 mL of 0.2% (wt/vol) pyrogallol dissolved in methanol and vortex for 3 minutes.
• Add 1.25 mL of 60% (wt/vol) KOH and vortex for 10 seconds.
• Incubate for 1 h at 75ºC, vortexing every 15 minutes.
• Add appropriate amount of hexane.
• Centrifuge tubes for 5 minutes at 2,800 rpm.
• Pipette 1 mL of hexane into a cuvette.
• Measure absorbance at 449nm.
• Total carotenoids (in µg/g[dw]) = (A449 x mL hexane) / (0.2702 x g [dw])

(Verwaal, R., Wang, J. Meijnen, J., Visser, H., Sandmann, G., van den Berg, J., van Ooyen, A. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied Environmental Microbiology 2007.)

L-Ascorbic Acid Assay

• Inoculate cells at an initial optical density of 0.05 medium and grow for about 18 hours.
• Spin down in centrifuge at 4000 rpm for 5 min at 4°C.
• Re-suspend in ice cold distilled water then spin down in centrifuge at 4000 rpm for 5 min at 4°C.
• Resuspended in about three times volume of ice-cold 10% (w/v) trichloroacetic acid, vortexed vigorously, and kept in ice for 20 minutes.
• Centrifuge and collect supernatant.
• Determine Ascorbic acid spectrophotometrically following a method adapted from that of Sullivan et Clarke: 35 μl of sample mixed in a cuvette with 40 μl of H3PO4 (85%), 675 μl of α,α′-bipyridyl (0.5%), and 135 μl of FeCl3 (1%), after 10 min at room temperature, the absorbance at 525 nm was measured.