Experiments started January 2016

We are starting a new series of experiments in 2016. The goal of these experiments is to provably express some quantity of full-length bovine kappa-casein (CSN3*B). Kappa-casein is the most important protein for cheese.

A bit of background: It's likely that all other proteins in cheese can be reasonably replaced with other vegan proteins, but kappa-casein has the unique property as acting for proteins in a watery solution as soap acts for oil in a watery solution: Kappa-casein forms micelles (little spheres) of protein, presenting a hydrophillic end to the water outside the micelle and a hydrophic end to the inside of the micelle. This allows large amounts of protein to be dissolved in a watery solution and when Chymosin (the primary active enzyme in rennet) is added, it cleaves the hydrophillic end from the kappa-casein which causes the protein to rapidly drop out of solution, entrapping many milk-fats in the process. The resulting chunk of semi-solid protein, fat and water is what we call cheese curds.

These experiments are based on the Kim2005 paper where a partial human kappa-casein was expressed in S. cerevisiae.

The experiments will be conducted in parallel at Counter Culture Labs in Oakland and BioCurious in Sunnyvale.

Briefly, we're using an E. coli / S. cerevisiae shuttle vector from DNA 2.0. We're using ampicillin for E. coli selection and Ura3 for yeast selection (with 5-FOA counter-selection) and we're using the GAL1 galactose-inducible promoter. We're also using an alpha-factor secretion sequence in an attempt to secrete the protein and a his tag to purify the protein.

Here is our:

• Experimental design
• Plasmid design
• List of stuff to purchase
• Raw Experimental Notes June 2016
• ProcessDiagram