Week 09 — Cell Free Systems

Date: Tue Apr 1. Slides: (will be posted when available) Recording: Zoom

Warning Mandatory for Committed Listeners and MIT/Harvard students. Part A — Questions Key readings: miniPCR — DNAdots: Cell‑free technology ACS Synth Biol (2024) Explain the main advantages of cell‑free protein synthesis over in vivo expression. Name two cases where cell‑free is preferable. Describe the main components of a cell‑free expression system and the role of each. Why is energy provision/regeneration critical? Propose a method to ensure a continuous ATP supply. Compare prokaryotic vs eukaryotic cell‑free systems. Choose a protein for each and justify. Design an experiment to optimize expression of a membrane protein; list challenges and mitigations. You observe low yield; provide three plausible causes and a troubleshooting plan for each. Part B — Individual Final Project Report Tip Start exploring your final project in depth this week. The minimum requirement is completing Aim 1.

Date: Wed Apr 2. Slides: will be shared here after class. Recording: Zoom

Info MIT/Harvard: No in‑person lab protocol this week (per schedule). Global Nodes: Use the PDF protocol below. Objective Induce a reporter in a cell‑free transcription‑translation (TX‑TL) system and quantify expression changes between treatments. Concepts You’ll Learn TX‑TL reaction components and setup Induction strategies and controls Fluorescence quantification & fold‑change analysis Protocol Global Labs Protocol (PDF) — Cell_Free_System_Laboratory_(Global_Labs).pdf Post‑Lab Questions (Mandatory) Using your lab images (or provided images), compute fluorescence fold‑change between treatments. Discuss LacI function in the context of the circuit used. What would you expect if the same induction were performed in E. coli BL21(DE3)?

miniPCR — DNAdots: Cell‑free technology — PDF Recent cell‑free system advances — ACS Synth Biol (2024) Example project gallery — Google Slide Deck