Week 06 — Genetic Circuits Part I

Date: Tue Mar 11. Slides: Chris Mason — “A 500‑year plan for engineering life on Earth and beyond” Lisa Riedmayr — “Genetic Medicines” Recording: Zoom

Warning These questions are based on the Gibson Assembly Lab and are mandatory for Committed Listeners and MIT/Harvard students. Key Link Gibson Assembly — Recitation/Protocol (Google Doc) Questions What are some components in the Phusion High‑Fidelity PCR Master Mix and what is their purpose? What are some factors that determine primer annealing temperature during PCR? There are two methods in this protocol that create linear fragments of DNA: PCR and restriction enzyme digest. Compare and contrast these two methods, including when one may be preferable. Why does the PvuII digest require CutSmart buffer? How can you ensure that the DNA sequences you digested and PCR‑ed will be appropriate for Gibson cloning? How does the plasmid DNA enter E. coli cells during transformation? Describe another assembly method in detail (e.g., Golden Gate Assembly) in 5–7 sentences with diagrams. Model this method with Benchling or a similar tool.

Date: Wed Mar 12. Slides: will be shared after the class. Recording: Zoom

Tip This week’s lab pairs directly with the Homework. Use the protocol and primer‑design notes below; document your process and results. Objective Practice DNA assembly by preparing linear fragments (via PCR and/or restriction digest) and assembling them with Gibson Assembly, followed by transformation into E. coli. Concepts You’ll Learn Primer design fundamentals PCR setup and thermocycling Restriction digests & buffer selection Gibson Assembly principles & workflow Transformation and selection Protocol & Prep Gibson Assembly (Google Doc) Primer Design — Supplemental to Gibson Assembly Recitation

Primer Design — Supplemental to Gibson Assembly Recitation Introduction to Gibson Assembly — YouTube More from NEB — NEB: Gibson Assembly