Miles Wilderman, Whitman College Junior Biology Major, shares about their internship at Fred Hutchinson Cancer Research Center, Seattle WA
“My project for the summer at Fred Hutchinson is to design chimeric proteins by splicing parts of Endophilin A1 BAR domain with the BAR domain of Endophilin B1. I will then test the function of these proteins in the nematode C. elegans. Endophilin A1 rescues the unc-57 C. elegans, while Endophilin B1 does not fully rescue the uncoordinated worms back to wildtype. My initial interest was if proteins with similar structure can have parts substituted into each other. While working on this project I have learned a lot more than I bargained for. From general worm maintenance to isothermal assembly, bacterial transformation, and performing micro injection on the worms, I have had to fully dive into life in a lab. I started out by using UCSF chimeraX software to run and visualize Alphafold’s protein structure predictions. A large part of creating new chimeric proteins is being able to tell if the new protein will fold correctly. If it can’t fold correctly, it will not be expressed correctly in the phenotype later. Alphafold is new machine learning code that can predict the structure of the protein based on the given amino acid sequence as well as color coding the protein based on the model’s certainty of the structure in that region. I understand some of the code thanks to Parteek Kumar’s machine learning class this last semester at Whitman College, however much of it is over my head. Once I have structures that work well and I believe I have enough and the right bits of Endophilin A1 to rescue the worms, I can take the amino acid sequence and convert it into
Wilderman 2 the corresponding dna sequence. I then use 
SnapGene software to import this dna sequence into the dna vector that is closest to what I want to be using for my protein. Then time for some pipetting. The lab orders synthetic dna for newly designed inserts, and I resuspend them with water and Tris. I ran a PCR to make many copies of this synthetic
dna. I had to remake the vector that I needed for my insert because the vector that I wanted was made by the lab many years ago. I found the glycerol stock for my vector where they freeze all plasmids in a -80C freezer. I regrew the bacteria, and purified out my plasmid. I then cut out the chuck of my plasmid that I wanted to change with enzymes, and added rSAP to dephosphorylate the ends of my cuts so that the vector didn’t ligate back together. Next came isothermal assembly where I added my synthetic dna insert to the vector. After isothermal, I transformed the plasmid into competent bacterial cells and heat shocked my plasmid into the bacteria. This allows me to grow more of the exact dna that I need if I ever run out later. Then purified out my final plasmid that I will ultimately inject into C. elegans. So far this summer, I have 5 plasmids containing the genetic code for my designed proteins ready for injection, and I am currently working on many more. This week with Monday being July 17, I will be injecting my designed proteins for the first time and hopefully I will save the poor unc-57 worms.”