Week 11: Building Genomes

Context
This week is about designing and assembling DNA at larger scales—from multi-kb constructs up to synthetic chromosomes—plus what “genome design” actually changes (recoding, tRNAs, loxPsym/SCRaMbLE, etc.). Good background reads are linked below.

Goals

  • Understand how DNA fragments are assembled into larger constructs (e.g., Gibson assembly). :contentReference[oaicite:0]{index=0}
  • See how minimal/synthetic genomes are built and why (e.g., JCVI-syn3.0, Sc2.0). :contentReference[oaicite:1]{index=1}
  • Explore genome-scale design choices: stop-codon recoding, moving tRNA genes to a neochromosome, and adding loxPsym sites for SCRaMbLE. :contentReference[oaicite:2]{index=2}

Background (skim these)

  • How to build a genome — overview of tools from gene → chromosome. :contentReference[oaicite:3]{index=3}
  • Building genomes to understand biology — perspective on synthetic genomics & what it enables. :contentReference[oaicite:4]{index=4}
  • Gibson Assembly (protocol & notes) — practical “how it works” refresher. :contentReference[oaicite:5]{index=5}
  • JCVI-syn3.0 minimal cell (Science 2016 + overview). :contentReference[oaicite:6]{index=6}
  • Sc2.0 (synthetic yeast genome): milestones, tRNA neochromosome, loxPsym/SCRaMbLE. :contentReference[oaicite:7]{index=7}

Part A — Plan a multi-fragment assembly (paper design)

Pick a 3–6 fragment construct (total 3–10 kb, your choice). Design it as if you’ll assemble with Gibson:

  1. Fragments & overlaps

    • Choose fragment boundaries and specify 20–40 bp overlaps compatible with Gibson.
    • Record lengths and GC%; avoid extreme GC in overlaps. :contentReference[oaicite:8]{index=8}

  2. Primer plan

    • Draft primer sequences that add each overlap (annotate 5′ overlap vs. 3′ gene-specific portions).

  3. Assembly notes

    • Note vector backbone, selection marker, and any features (origin, promoter, etc.).
    • Brief risk list (repeats, homopolymers, secondary structure).

Deliverables (put right on this page):

  • A small table (Fragment, Size (bp), Left overlap, Right overlap, Notes).
  • Your primers (5′→3′) with labeled overlap regions.
  • One paragraph explaining why you chose these cut points/overlaps. :contentReference[oaicite:9]{index=9}

Part B — Genome-scale design thought exercise

Pick one of these scenarios and write ~6–10 bullets + 1 short paragraph:

  • Recoding a stop codon (TAG→TAA) across a genome to free a codon for future use. Consider impacts on essential genes, synthesis/validation, and compatibility. Background on genome-scale builds helps frame the challenge. :contentReference[oaicite:10]{index=10}
  • Move all nuclear tRNA genes to a tRNA “neochromosome”. Why do this? What breaks if you don’t (stability, hotspots), and what checks would you run? :contentReference[oaicite:11]{index=11}
  • Install loxPsym sites (every ~10 kb or at 3′ ends of non-essential genes) to enable SCRaMbLE for rapid in-vivo rearrangement. What are the safety/containment and debugging concerns? :contentReference[oaicite:12]{index=12}

Deliverables:

  • Your chosen scenario, concise bullet plan, and a 1-paragraph risk/benefit summary with 1–2 citations. :contentReference[oaicite:13]{index=13}

Part C — Case studies (pick one to summarize)

  • Minimal cell JCVI-syn3.0: what remained, what functions were unknown, and why a minimal chassis matters. :contentReference[oaicite:14]{index=14}
  • Sc2.0 consolidation: progress, issues uncovered (e.g., growth defects), and how debugging fixed them; summarize a figure/result you find compelling. :contentReference[oaicite:15]{index=15}

Deliverable: 6–8 sentence summary + one “design lesson” you’d reuse.

Submission checklist

  • Part A: assembly table + primers + design notes (Gibson-ready). :contentReference[oaicite:16]{index=16}
  • Part B: genome-scale scenario bullets + short risk/benefit paragraph with citations. :contentReference[oaicite:17]{index=17}
  • Part C: case-study summary + one “design lesson.” :contentReference[oaicite:18]{index=18}
  • Nature “How to build a genome.” :contentReference[oaicite:19]{index=19}
  • Nat. Commun. “Building genomes to understand biology.” (PDF) :contentReference[oaicite:20]{index=20}
  • Addgene Gibson Assembly protocol. :contentReference[oaicite:21]{index=21}
  • JCVI-syn3.0 minimal genome (Science + overview). :contentReference[oaicite:22]{index=22}
  • Sc2.0: tRNA neochromosome; loxPsym/SCRaMbLE overview & papers. :contentReference[oaicite:23]{index=23}