Week 9: Cell Free Systems

Context
Week 9 explores cell-free protein expression (TX–TL) and why it’s useful for prototyping, biosensing, and making proteins on demand. Page structure mirrors your original notes. :contentReference[oaicite:0]{index=0}

Timetable (work log)

Lecture (4/1)

• Class recording: https://mit.zoom.us/rec/share/0by8nzXE7HNoPPim_6gNzeb6k4QRyU1H_0Vo-6GD5YwPqM3vkUiDJeHNqacYrBVo.mUfLX6vRH92zK7xD :contentReference[oaicite:1]{index=1}
• Slides will be shared here after class.

Recitation (4/2)

• Recitation recording: https://mit.zoom.us/rec/share/mDl9azWFrjSKm_CXE37GAShHn4LJXhBQVSVEwNesjwEOYEE0-1ptN8wLEZpc5O05.fiDj0_EUcO8-szP_ :contentReference[oaicite:2]{index=2}
• Slides link will be added here after class.

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Homework — Part A

Prompt:
Explain the main advantages of cell-free protein synthesis over in vivo expression, focusing on flexibility and control. Name at least two cases where cell-free is preferable to cell-based production. :contentReference[oaicite:4]{index=4}

My short answer

• Faster start-to-data: no culturing required; reactions can be set up directly with DNA template + TX–TL mix. :contentReference[oaicite:5]{index=5}
• Open & controllable chemistry: total control over DNA concentration, energy system, amino acids, cofactors, salts, and additives—without cell-membrane barriers or endogenous regulation. :contentReference[oaicite:6]{index=6}
• Safer for the host / tolerant of toxic payloads: express proteins that would kill or stress living hosts (toxins, membrane proteins, lytic peptides). :contentReference[oaicite:7]{index=7}
• Convenient for unusual chemistry: easier incorporation of unnatural amino acids or non-standard components by spiking the reaction. :contentReference[oaicite:8]{index=8}

When cell-free wins (examples):

1. Toxic proteins (e.g., pore-formers, nucleases) that crash cell cultures. :contentReference[oaicite:9]{index=9}
2. Point-of-need / on-demand production (field diagnostics, small-batch enzymes, education kits). :contentReference[oaicite:10]{index=10}

What’s inside a cell-free reaction (at a glance)

• DNA/RNA template (what to express)
• Transcription/translation machinery (from extract or reconstituted system)
• Amino acids + nucleotides
• Energy system (ATP regeneration) and cofactors
  These components are combined in an “open” reaction, enabling direct tuning and additions. :contentReference[oaicite:11]{index=11}

Submission checklist

• A concise answer covering advantages (speed, control, openness)
• Two cases where cell-free is preferable (e.g., toxic proteins; on-demand production/field use)
• One paragraph on what you would prototype with TX–TL next (sensor, reporter, enzyme)

References

• miniPCR DNAdots — Cell-free technology (primer & components). :contentReference[oaicite:14]{index=14}
• CFPS applied “at point of need” (ISS validation paper). :contentReference[oaicite:15]{index=15}
• Overview article on when to use cell-free (pros/cons & system choices). :contentReference[oaicite:16]{index=16}