cell-free-systems

General Homework Questions

  1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production.
  • One advantage includes the fact that cell-free protein synthesis gives direct control over reaction composition which means we can precisely control the exact concenctrations of factors like DNA, amino acids and modified nucleotides.
  • No cell membrane barrier means everything is immediately accessible and modifiable
  • The speed at which you can cycle designs and tests is much faster thant that if you’d have to do the usual process of cloning then transform then grow
  • You can precisely and exactly control the specific environments its in, setting the exact pH, temperature and also be able to eliminate toxic constraints.

  1. The bread and butter for cell free systems is the cell extract which includes the ribosomes that are the machinery for protein synthesis, tRNAs to deliver amino acids, translation factors and also the enzymes required for and involved in metabolism.

  2. Energy regeneration is critical in cell-free protein synthesis because ATP is rapidly consumed during transcription and translation, and unlike in living cells, there are no metabolic pathways to replenish it, causing protein synthesis to quickly stop if energy is depleted. Simply adding ATP is insufficient due to rapid consumption and accumulation of inhibitory byproducts, so regeneration systems are required to sustain reactions and improve yield. One common method is the phosphoenolpyruvate (PEP) system, in which PEP donates a phosphate group to ADP via enzymes such as pyruvate kinase present in extracts from Escherichia coli, continuously regenerating ATP and allowing protein synthesis to proceed for longer durations.

  3. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

  • Prokaryotic cell-free systems, typically derived from Escherichia coli, are fast, cost-effective, and produce high yields, but lack the machinery for complex post-translational modifications such as glycosylation or proper disulfide bond formation.

  • In contrast, eukaryotic systems (e.g., wheat germ or rabbit reticulocyte extracts) are slower and more expensive but support proper folding and modifications required for many eukaryotic proteins.

  • For example, a simple enzyme such as Green Fluorescent Protein can be efficiently produced in a prokaryotic system because it does not require extensive post-translational modifications, making it ideal for rapid, high-yield expression. Conversely, a therapeutic protein like Insulin is better suited to a eukaryotic system, as it requires correct folding and disulfide bond formation to be biologically active.

  1. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup
  • To optimise expression of a membrane protein in a cell-free system, I would design the experiment as a small screening setup in which the same DNA template is expressed across multiple reaction conditions while varying factors that most strongly affect membrane protein yield and solubility
  1. This could most likely be due to a low concentration of the DNA fragment thats coding for the target protein we can fxi this by increasing the DNA fragment concentration

this could also be due to an enzyme thats accidentally been released into our cell free system thats cleaving our target protein and so we can fix this by checking and ensuring no said enzymes are produced.

This could also be due to a lack of amino acid building blocks to actually synthesise the protein and so we can add a better nutrient medium to ensure this is not in limiting.

Kate Adamala’s HW

  1. My synthetic minimal cell would be a sense-and-respond therapeutic cell that detects joint degradation, the input would be lower levels of cartilage or even higher levels of inflammation in those regions and the output would be release of chemicals that provide lubrication around the said joint.

It could probably not be realised by cell-free Tx/TI alone.

Yes, a genetically modified natural cell would make more sense as it would be able to effectively respond within the organisms cells.

A mammalian cell-free system (HeLa or CHO cell extract is most common) is probably necessary here, primarily because:

  • Therapeutic protein functionality depends on correct PTMs
  • NF-κB or similar mammalian response elements are the natural way to sense the arthritic environment
  • Immunogenicity in the joint space makes bacterial components a serious liability

The desired outcome would be that during times of discomfort or too much inflammation in joints for those with arthritis, the synthetic cell operation would allow to alleviate for some of this.

Peter Nguyen’s homework questions:

One-sentence pitch A building wall system embedded with freeze-dried cell-free biosensors that detect seasonal temperature shifts and autonomously trigger the germination of different plant species — turning architecture into a living calendar.

How it works The wall panels contain a layered substrate of freeze-dried cell-free systems encapsulated in a hydrogel matrix alongside dormant seeds from different plant species. Each CFS unit is engineered with a temperature-sensitive riboswitch or cold-shock promoter tuned to a specific threshold — say, sub-10°C for winter species, 18–22°C for spring/summer. When the trigger temperature is reached, the CFS activates, expressing germination-promoting enzymes (e.g. gibberellin-related proteins or cell-wall loosening factors) that are secreted locally into the seed microenvironment, initiating growth of that season’s designated plant. Different zones of the wall cycle through moss, herbs, climbing flowers, or ornamental grasses depending on the external climate — with no human input.

Societal challenge / market need Urban spaces are increasingly disconnected from natural seasonal rhythms, contributing to biophilic deficit in dense cities. The indoor plant and living wall market is growing rapidly but remains static — the same wall looks identical year-round. This system makes buildings genuinely responsive organisms, with applications in hospitality, retail, wellbeing spaces, and sustainable architecture.

Addressing CFS limitations The hydrogel encapsulation keeps the freeze-dried CFS stable and dormant until ambient humidity or a deliberate water-activation event (rainfall ingress, irrigation pulse) rehydrates the matrix. The one-time-use constraint is turned into a feature — each CFS pod is a single-season unit, designed to be swapped out in modular panel cartridges at the start of each season, making maintenance a ritual rather than a problem. Long-term stability is extended by storing unactivated pods at the panel’s rear in a desiccated micro-chamber until the thermal trigger is met.

Ally Huang’s homework Questions

  1. In outer space exploration the most VITAL step and building of infrastructure has to be initializing sustainable crop growth to not only kick-start O2 production to make the new planets/area habitable but to also allow for one of the most underlooked factors which is aggriculture! Since its a given in the world we live in now, we underestimate that itll take alot to produce fertile plants that produce edible crops to allow for common food to be produced later into space exploration.

  2. Nitrogenase complex genes (nifH, nifD, nifK) and ATP-regeneration pathways, engineered into a cell-free synthetic biology platform for on-site biological nitrogen fixation and soil fertilization in extraterrestrial agricultural systems.

  3. Nitrogen is the most limiting nutrient for crop production, yet no extraterrestrial environment has biologically available nitrogen in soil. The nitrogenase complex genes (nifH, nifD, nifK) encode the only known enzymatic machinery capable of converting atmospheric N₂ into bioavailable ammonia — the foundation of all plant protein and growth. Deploying these genes within a cell-free platform eliminates dependence on Earth-supplied fertilizers, removes the need for living microorganisms that pose contamination risks in sterile extraterrestrial soils, and enables programmable, on-demand nutrient production. Solving this bottleneck directly unlocks sustainable crop growth, oxygen generation, and long-term human habitability beyond Earth.

  4. We hypothesize that a cell-free synthetic biology system expressing nifH, nifD, and nifK with an integrated ATP-regeneration cascade can fix atmospheric N₂ into bioavailable ammonia at rates sufficient to fertilize extraterrestrial soil simulants, enabling measurable crop germination and growth without Earth-supplied nitrogen fertilizers — establishing a foundational, contamination-free nutrient production platform for space agriculture.

  5. Experimental Plan: Samples: Cell-free reactions with purified nitrogenase complex tested against Mars/lunar soil simulants, with and without ATP-regeneration systems. Controls: Boiled/inactive enzyme controls, Earth soil with standard fertilizer, and unfertilized simulant. Measurements: Ammonia output (colorimetric assay), plant germination rates, biomass yield, chlorophyll content, and O₂ production over 30-day growth trials.