Team:RUBochum/Proof Of Concept
Proof of concept
Our goal was to produce platypus yogurt with tasty ingredients.
During our short lab time, we created 5 out of 12 bioinformatics-based constructs which have been successfully transformed into P. pastoris.
Figure 1: Example of P. pastoris transformants: 7. Brazzein, 8. β-casein bos taurus.
We also started the overexpression of the proteins.
Figure 2: NanoDrop measurement of the 72h supernatant. Ordered from right to left: 1. αs1 casein Bos taurus (26,3 mg/ml), 2. Brazzein (69,9 mg/ml), 3. β casein Ornithorhynchus anatinus (43,6 mg/ml)
The samples were purified by size. Measured with the respective molecular weight and molar attenuation coefficient the protein yield was determined αs1 casein Bos taurus (26,3 mg/ml), Brazzein (69,9 mg/ml), β casein Ornithorhynchus anatinus (43,6 mg/ml).
Due to the test measurements and rough estimations, we expect a minimum protein yield of around 20 mg/ml. Related to the concentration of αS1-Casein 10 g/l in bovine milk, it would require half a liter of culture to produce one liter of milk. In contrast, one liter milk of common dairy cows requires 545 l of water.
Figure 3: Water needed for on 1l Milk: Conventional dairy farming 545 l, RUBochums synthetic milk 1,5 l .
Proteins couldn't be detected on the SDS-page due to an unknown error source which might be related to the staining solution.
With further optimization and chromosomal integrations, purification would be simplified due to the absence of selection markers.
With the elaborated heterologous fermentation step and its optimization, processes could get more efficient and easier.
Summary and Outlook