https://pages.htgaa.org/2026b/nitin-vashisht/week-03-hw-cell-free/index.htmlHTBAA Homework #3: Designing a Cell-Free Reaction Composition Prepared by: Nitin Due: Tuesday, 6 October 2026 0. Scope, and how this relates to my main project This is a deliberate branch off my HW1/HW2 thread rather than a continuation of it. My main project is a lab-in-the-loop system for discovering aptamer biosensors, and in HW2 I designed a theophylline-riboswitch-gated sfGFP construct to validate the cell-free layer of that pipeline. HW3 fixes the three protein targets (sfGFP, PETase, Reteplase) and fixes the reporter sequence, so the riboswitch design does not transfer directly. What does transfer is the reaction chemistry underneath it: every assay in my pipeline runs in an E. coli lysate CFPS reaction, so a cheaper and more reproducible reaction composition lowers the per-assay cost of my entire HW1 scale estimate. I return to that connection briefly in Section D.Hugoen