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Future Plans
In the future, we plan to characterize our system by growing liquid cultures and assaying samples for ascorbic acid at incremental time intervals to establish rate constants as well as quantify how much vitamin C (ascorbic acid) our system can produce.
Once we have Vitamin C over time curves we will run a comparative study on how yeast producing Vitamin C grows on YPD plates (a lab standard) versus how it grows on our desired substrate, bread. To test its growth on bread we have created plates of dough like media.
To optimize the production of ascorbic acid on the new substrate we will apply a strategy of directed evolution. We will make a combinatorial library of expression cassettes using golden gate assembly for every synthesized orf in the Vitamin C pathway. This will be made using promoters and terminators of varying strengths from across the genome. Once constructed, this library will be plated on dough media plate with a calculated amount of peroxide in the agar. The presence of the peroxide will apply oxidative stress to the cells and as Vitamin C confers oxidative stress resistance this behaves as a selection for the cells that producing more Vitamin C. By adjusting the amount of peroxide on the plate we can create a threshold of ascorbic acid production below which all the cells will die. To establish which one of the library members that passed through the selection is the best, we will perform a quantitative screen on the survivors. This will be done by growing up the cells in liquid culture and then extracting the ascorbic acid and spectrophotomertically determining its concentration.
