Originally, we wanted to clone the gene responsible for making the monarch butterfly taste bad to birds and/or make them sick. We were unable to find any genes responsible for this, since they actually sequester normally toxic chemicals from the milkweed plant. The plants produce these chemicals (known as cardenolides) via a biochemical pathway of which the enzyme progesterone 5-beta reductase is a part. This enzyme metabolizes progesterone into 5-beta-pregnane-3,20-dione, which is a steroid metabolite which we will test for using high performance liquid chromatography. Since there is no recorded gene sequence for this enzyme in the milkweed plant, we will be using a gene from a species of spike moss. We plan to order our Selaginella moellendorffii online from Plant Delights Nursery. Our gene sequence was located in the NCBI GenBank. Its accession number is NW_003314261. It is 1185 base pairs in length, and contains no introns. There is one BioBrick enzyme restriction site in our gene, so we will be using site-directed mutagenesis to change one nucleotide. Since the restriction site is near the beginning of our gene, we will include the mutated nucleotide in our primer used in PCR. We will not be attempting to add the BioBrick prefix to the forward primer, because it would be too long.
Reference article: Evolution of steroid-5-alpha-reductases in comparison of their function with 5ß-reductase DOI: 10.1016/j.ygcen.2009.08.004