week-09-hw-cell-free-systems

Homework question from Kate Adamala.

Design an example of a useful synthetic minimal cell as follows:

  1. Pick a function and describe it.

a. What would your synthetic cell do? What is the input, and what is the output?

  • The cell-free genetic circuit that I plan to make for the final project aims to detect different biological signals and produce a measurable output. The input will be one among the environmental signals, IL-6 or low O₂, and the output will be a green fluorescence signal or a therapeutic peptide.
  1. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
  • The system that I am thinking of needs to be encapsulated inside a hydrogel.
  1. Could this function be realized by genetically modified natural cells?
  • Cells do have a mechanism to respond to real signals in the body, but getting therapeutic peptides and other luminescent signals as an output from a signal is achieved if the cell is preprogrammed and the genetic circuit is assembled in a way to detect the signal and respond accordingly.
  1. Describe the desired outcome of your synthetic cell operation.
  • Output will be a Green Fluorescence Signal.

Design all components that would need to be part of your synthetic cell.

  1. What would the membrane be made of?

  2. What would you encapsulate inside? Enzymes, small molecules.

    • It will be encapsulated with hydrogel.

  3. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (Hint: for example, if you want to use small molecule modulated promoters, like Tet-ON, you need mammalian)

  4. How will your synthetic cell communicate with the environment? (Hint: are substrates permeable? Or do you need to express the membrane channel?)

Experimental details

List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel,” pick the actual gene.) How will you measure the function of your system?

Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material.

Answer each of these key questions for your proposal pitch:

  1. Write a one-sentence summary pitch sentence describing your concept.

    • Synthetic fermenters that can be used in the preparation of household fermented drinks and foods.

  2. How will the idea work, in more detail? Write 3-4 sentences or more.

    • A yeast-inspired fermenter that can be used all year round, when making different fermented foods and drinks.

  3. What societal challenge or market need will this address?

    • It would decrease the dependency on natural yeast that may not be available, and it may also not be as functional as it should be.

  4. How do you envision addressing the limitations of cell-free reactions (e.g., activation with water, stability, one-time use)?

    • Freeze-Drying (Lyophilizing)

Homework question from Ally Huang

Genes in Space Proposal: On-Demand Drug Synthesis Using Freeze-Dried Cell-Free Systems

  1. Background & Significance.

  • During long-duration spaceflight, astronauts face unique medical challenges, including immune dysregulation, increased infection susceptibility, and limited access to pharmaceuticals. Resupply missions from Earth take months, and stored drugs degrade under radiation and temperature fluctuations aboard the ISS. This creates a critical gap in astronaut healthcare. Freeze-dried (lyophilized) cell-free protein synthesis (CFPS) systems offer a revolutionary solution: producing therapeutic proteins on-demand without live cells or complex equipment. Developing this capability is significant for humanity as it could also enable point-of-care drug manufacturing in remote locations on Earth, while advancing our understanding of biochemical reactions in microgravity.

  1. Target: The human granulocyte-colony stimulating factor (hG-CSF) gene — a therapeutic protein that stimulates white blood cell production, critical for combating spaceflight-induced immune suppression.

  2. Relationship to the Space Biology Challenge.

Spaceflight significantly suppresses immune function, leaving astronauts vulnerable to opportunistic infections with no immediate access to Earth-based medical support. hG-CSF is a clinically proven immunostimulatory cytokine used on Earth to treat neutropenia (low white blood cell count). By encoding the hG-CSF gene in a DNA template compatible with a freeze-dried CFPS system, we can synthesize this therapeutic protein directly aboard the ISS when needed. This eliminates reliance on pre-packaged drugs that degrade over time and demonstrates that biologically active therapeutics can be manufactured in a microgravity environment using minimal, shelf-stable reagents.

  1. Hypothesis & Reasoning.

Hypothesis: A freeze-dried cell-free protein synthesis system can successfully express biologically active hG-CSF protein aboard the ISS, with yields and activity comparable to Earth-based controls. We reason that CFPS systems, which contain all necessary transcription and translation machinery extracted from bacterial cells and lyophilized for stability, should retain functionality in microgravity, as the core biochemical reactions are molecular in nature and do not inherently depend on gravity. However, microgravity may alter fluid dynamics, molecular diffusion, and reaction kinetics in ways that affect protein folding and yield. By comparing ISS-synthesized hG-CSF to ground controls using the same freeze-dried BioBits platform, we can directly quantify any performance differences. If successful, this establishes a proof-of-concept for in-space pharmaceutical biomanufacturing, paving the way for astronauts on deep-space missions to synthesize a broad library of therapeutics from compact, stable DNA templates.

  1. Experimental Plan.

Samples: Freeze-dried BioBits CFPS extract rehydrated with a plasmid encoding hG-CSF, tested aboard the ISS and in a matched ground control. Controls:

  • Negative control: CFPS extract rehydrated without plasmid DNA
  • Positive control: Ground-based hG-CSF expression under identical conditions

Measurements:

Protein yield quantified via fluorescent reporter tag (GFP-fused hG-CSF) using the Genes in Space Fluorescence Viewer Reaction kinetics tracked at 30-minute intervals over 4 hours Biological activity assessed post-flight via cell proliferation assay

Data will be compared between ISS and ground samples to evaluate the effect of microgravity on CFPS efficiency and protein functionality.