Week 09 HW: CELL FREE SYSTEMS

Part One: General and Lecturer-Specific Questions 1.

Cell-free protein synthesis gives better control over reaction conditions and allows direct addition of molecules like inhibitors or non-natural amino acids. It is useful for producing toxic proteins and for rapid protein prototyping.

A cell-free system contains lysate with ribosomes and enzymes, a DNA/mRNA template, amino acids, and buffer salts. These components work together to produce proteins from genetic instructions.

ATP regeneration is important because protein synthesis uses large amounts of energy. A creatine phosphate–creatine kinase system can recycle ADP into ATP and maintain continuous protein production.

Prokaryotic systems are fast and cheap, making them suitable for simple proteins like GFP. Eukaryotic systems are better for complex proteins like antibodies because they support proper folding and modifications.

Membrane proteins need lipid structures like liposomes or nanodiscs to fold correctly. The main challenge is balancing detergent levels to prevent aggregation without damaging the system.

Low protein yield may result from poor DNA quality, RNase contamination, or incorrect magnesium concentration. These can be fixed by purifying DNA, adding RNase inhibitors, and optimizing salt levels.

Part two :

Kate Adamala - Synthetic Minimal Cell

Function: Endometriosis pain relief on demand

A synthetic minimal cell that senses elevated prostaglandin E2 (a key inflammatory marker in endometriosis) and responds by synthesizing and releasing ibuprofen precursor enzymes locally within the peritoneal cavity. Unlike systemic NSAIDs, this SMC delivers anti-inflammatory action only where inflammation is detected, reducing systemic side effects.

Components:

  • POPC/cholesterol lipid bilayer for stability in peritoneal fluid
  • PURE system encapsulated inside
  • α-hemolysin nanopores for prostaglandin sensing and enzyme secretion
  • pPGE2-responsive promoter driving COX-2 inhibitor peptide synthesis
  • sfGFP reporter fused to output peptide for activity confirmation

Experimental validation:

  • Fluorometry to confirm GFP-tagged peptide production upon PGE2 stimulation
  • ELISA to measure prostaglandin reduction in treated vs untreated samples
  • Cytotoxicity assay on peritoneal cell lines to confirm biocompatibility

Peter Nguyen - Architectural Textile

Pitch: A wearable menstrual health textile that bioluminesces to signal the onset of infection or abnormal pH associated with bacterial vaginosis or endometriosis flares, requiring no battery or external device.

Mechanism:

  • Porous underwear-lining mesh embedded with microcapsules containing freeze-dried TX-TL components
  • DNA circuit triggered by vaginal pH drop below 4.0 or presence of Gardnerella vaginalis enzyme sialidase
  • Upon trigger, capsules absorb local moisture and translate luciferase reporter, producing visible bioluminescence at infection site
  • Lyoprotectant matrix keeps enzymes stable for 12 months dry storage
  • Rechargeable via buffer spray; signal resets once pH normalises

Societal challenge addressed: Bacterial vaginosis affects 1 in 3 women globally and is frequently misdiagnosed or ignored. A passive, wearable biosensor democratises early detection without clinic access.

Ally Huang - Cell-Free BioBits

Target: On-demand psilocybin biosynthesis for treatment-resistant depression in isolated or underserved communities

Women and queer individuals experience depression at disproportionately higher rates yet have less access to emerging psychedelic-assisted therapies due to legal, geographic, and financial barriers. A cell-free system producing therapeutic-dose psilocybin on demand from a freeze-dried DNA template offers a decentralised, stable, and scalable alternative to clinic-dependent treatment.

Genetic target: Synthetic DNA encoding the four-enzyme psilocybin biosynthesis pathway (PsiD, PsiK, PsiM, PsiH) from Psilocybe cubensis, codon-optimised for BioBits cell-free expression.

Hypothesis: The BioBits freeze-dried matrix will support sequential multi-enzyme expression sufficient to produce measurable psilocybin from tryptamine substrate at 37°C without living cells.

Experiment:

  • Three conditions: GFP positive control, four-enzyme pathway test, no-DNA negative control
  • Activation with water + tryptamine substrate
  • Output measured via LC-MS for psilocybin quantification and P51 viewer for fluorescent pathway reporter
  • Success = detectable psilocybin above therapeutic threshold (1–3mg equivalent) per reaction volume

Why cell-free: No living GMO organism, no containment risk, stable as freeze-dried powder for field or home use, legally easier to research than whole-cell biosynthesis.

Have used Claude (Sonnet 4.6 ) to brainstorm.