Experiments
Experiment 0
Description in progress
Experiment 1
Experiment 1 will use well-studied methods previously tested in Y. lipolytica. We will use Gibson cloning to assemble our five genes with our HAs into the linearized pUC19 plasmid. We will amplify this engineered plasmid in E. coli and then isolate the plasmids. We will linearize them using the restriction enzyme Sma1, which cuts the plasmid in the multiple cloning sites between the HAs, and then transform Y. lipolytica using HR. We will select for our transformed yeast on 5-Fluoroorotic Acid (5-FOA) enriched with URA3 to select for cells who have successfully exchanged the URA3 gene between the HAs for our gene insert.
Experiment 2
Description in progress
Experiment 3
Description in progress
Quantification
Description in progress