College
College of Engineering
Mentor Information
Lawrence Stern
Description
CAR T cells have shown success in treating B cell malignancies but have shown little effectiveness in treating other cancers. Recently, the field has looked to cytokine signaling to overcome the suppressive signals they encounter in solid tumor microenvironments by incorporating key intracellular domains into the CAR structure. Our goal is to engineer and study JAK/STAT recruitment motifs in the intercellular domain of one such CAR. To accomplish this, I used recombinant DNA technology to combine the target JAK gene with a carrier DNA vector containing STAT, then transformed the resulting plasmid into e coli to produce large amounts of this plasmid containing both JAK1 and STAT3. This plasmid was then transformed into yeast for the purpose of Yeast Surface Display in which the protein of interest is phosphorylated in the endoplasmic reticulum and displayed on the cell wall of the yeast. Flow cytometry is then used to confirm successful phosphorylation and yeast surface display. We hope to find a population of cells which have STAT3 expressed on the surface with phosphorylation by JAK1. This data will create a positive control that will be vital for proof of concept in the lab moving forward.
Using Yeast Endoplasmic Reticulum Sequestration and Yeast Surface Display to Examine Expression and Phosphorylation of STAT3 by JAK1
CAR T cells have shown success in treating B cell malignancies but have shown little effectiveness in treating other cancers. Recently, the field has looked to cytokine signaling to overcome the suppressive signals they encounter in solid tumor microenvironments by incorporating key intracellular domains into the CAR structure. Our goal is to engineer and study JAK/STAT recruitment motifs in the intercellular domain of one such CAR. To accomplish this, I used recombinant DNA technology to combine the target JAK gene with a carrier DNA vector containing STAT, then transformed the resulting plasmid into e coli to produce large amounts of this plasmid containing both JAK1 and STAT3. This plasmid was then transformed into yeast for the purpose of Yeast Surface Display in which the protein of interest is phosphorylated in the endoplasmic reticulum and displayed on the cell wall of the yeast. Flow cytometry is then used to confirm successful phosphorylation and yeast surface display. We hope to find a population of cells which have STAT3 expressed on the surface with phosphorylation by JAK1. This data will create a positive control that will be vital for proof of concept in the lab moving forward.
