Week 11: Building Genomes
Cell-Free Protein Synthesis (CFPS)
CFPS recreates transcription and translation outside a living cell using a lysate supplemented with all the molecular machinery needed to produce a protein from a DNA template. No cell wall means direct control over every component.
The Lysate
The BL21(DE3) Star lysate provides ribosomes, tRNAs, elongation factors — and crucially, T7 RNA Polymerase, which transcribes any gene under a T7 promoter with high speed and efficiency.
Salts & Buffer
Maintain the ionic environment enzymes need. HEPES-KOH keeps pH ~7.5 (neutral, like inside a cell). Mg²⁺ supports ribosome assembly and polymerase activity. K⁺ is a co-factor for many enzymes. Potassium phosphate (mono + dibasic together) provides both phosphate for energy transfer and secondary buffering.
Energy & Nucleotides
AMP, CMP, GMP, UMP are the RNA building blocks. They’re monophosphates, so they must be phosphorylated to NTPs before incorporation into RNA. Ribose and glucose fuel the metabolic pathways that do this phosphorylation. Guanine is the free base for GMP — the cell’s salvage enzymes assemble it from guanine + ribose + phosphate, which is why GMP doesn’t need to be added directly.
Two Master Mix Strategies
| 1-hr PEP-NTP | 20-hr NMP-Ribose-Glucose | |
|---|---|---|
| Nucleotides | NTPs (ready to use) | NMPs (must be phosphorylated) |
| Energy | PEP (fast, direct phosphate donor) | Glycolysis-like pathways (slow, sustained) |
| Trade-off | Fast but expensive | Cheap but slow |
Amino Acids
All 20 amino acids must be present. Tyrosine and cysteine are added separately because tyrosine is poorly soluble and cysteine oxidizes easily — both would degrade in a bulk mix. Nicotinamide feeds NAD⁺/NADP⁺ production for redox-based energy regeneration.
Fluorescent Proteins in CFPS
The key properties that matter in a cell-free context:
- sfGFP — engineered for fast, robust folding; signal appears quickly
- mRFP1 — slow-maturing; delayed fluorescence signal
- mKO2 — oxygen-dependent maturation; limited O₂ in tubes is a bottleneck
- mTurquoise2 — high quantum yield, fast maturation; well-suited for CFPS
- mScarlet-I — brighter and faster-maturing than mRFP1
- Electra2 — newer, less characterized
Cloud Labs
Fully automated, remotely accessible labs where robots (liquid handlers, plate readers, incubators) execute experiments designed digitally. Key benefits: sub-microliter pipetting precision, massive parallelism (e.g. 1,536-well plates), global accessibility, and compatibility with AI-driven optimization loops.