Team:Johns Hopkins/Project/MeasureQuant

Measurements and Quantification

In order to assess the efficiency and effectiveness of our yeast strains, we performed a series of quantitative measurements which would allow us to determine protein expression levels, amount of products and bi-products synthesized, and kinetic rates of the enzymes in our system. The results of these experiments also provide important initial conditions for the modeling aspect of our project, as it provides us with reliable and realistic //in vivo// gene expression and catalytic activity of the enzymes specific to our system. The following experiments will allow us to quantitatively determine these desired expression/synthesis in our system.

HPLC for measuring product synthesis

• Measure the amount of products (β-carotene, L-Ascorbic Acid).

Protein Purification for enzyme assay

• Purify expressed protein of interest.
  1. PCR gene of interest-TEV-his6X-linker-mCherry-KAN. Tag is on C-terminus of the protein.
  2. Transform PCR products into yeast strains.
  3. Microscopy to show red fluorescence.
  4. Genomic prep from transformed cells and testing by PCR for integration of the tag at the c-terminus of the genes.
  5. Make a cell lysate and do a western with anti-his6x antibody to test for expression (both size and quantity).
  6. Flow cytometry to supplement expression quantity.

• • Enzyme Assay**
• Measurements to calculate the Km, kcat, and Vmax of the enzymes.