https://pages.htgaa.org/2026b/jamie-dixon/week-02-hw-dna-design/index.htmlOverview The steps to create a CBB resistant coffee tree Does not work: altering the genome of the tree using CRISPR of the tree b/c no one know how to do it - what site expresses the cherries for example What will works: altering the genome of the tree using a protein that destroys the gut of the CBB 1) Get the cry10Aa sequence and design the full expression cassette. 2) Order the synthesized cassette from Twist, cloned into your binary vector. 3) Amplify the plasmid in E. coli and verify it. 4) Transform Agrobacterium with the purified plasmid. 5) Co-culture with coffee somatic embryos, then select on antibiotic. 6) Regenerate plants, confirm the insert (PCR or Southern blot), and bioassay against the borer. Why the cry10Aa sequeence works 1) The insect eats the protein. A borer chewing into the berry ingests the toxin along with plant tissue. 2) The gut activates it. Insect midguts are typically alkaline and full of proteases, which convert the protein into its active form. 3) It binds gut receptors. The active toxin attaches to specific receptors on the midgut lining cells. The article notes that Cry proteins work this way, and that it's why organisms lacking the right receptors aren't affected. 4) The cells rupture. Binding leads to pores forming in the membrane, and the gut cells burst. 5) The insect dies. The damaged gut lets gut contents and bacteria leak into the body cavity, causing septicemia. What molecule am I expressing?Hugoen