Cell-Free Systems & Sensor Assignment

<aside> ⚠️ Mandatory for MIT/Harvard Students and Committed Listeners Due at the start of class April 1

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Read Cell-Free Protein Synthesis Primer- https://dnadots.minipcr.com/wp-content/uploads/2019/09/DNAdots-Cell-Free-Tech-final_qnoa.pdf by Ally Huang.

Answer 4 of following questions

• What is the advantage of encapsulating cell-free system into a liposome, making a synthetic cell? What is an example of application that you could not do with bulk cell-free system, but it becomes possible if the same translation system is encapsulated inside a membrane.

  • This allows to simulate organelles or cells in a better way, creating a barrier with the surrounding environment. For example, if you wanted to make an experiment to determine the passage of a protein or compound through the lipidic bilayer, unencapsulated systems wouldn´t allow you to determine the traffic through the barrier.

  

• Even though cell-free reactions do not contain cells, they do need to contain several essential components so proteins can still be expressed. List at least 5 biological components that a cell-free reaction needs (either extracted from living cells or supplemented by the scientists).

  • Cell-free reactions need the minimal biochemical components needed for protein synthesis (transcription and translation). This are:
    • Ribosomes
    • RNA polymerase
    • Nucleotids
    • tRNAs
    • Amino Acids
    • Energy in the form of ATP
    • Gene encoding the desired protein

• What are 2 similarities between the two methods of creating cell-free reactions? What are two differences?What advantages does freeze-drying the cell-free reaction have over just the cell-free reaction alone?

  • In both cases, all the components need to be added from previous purifications, and this allows a better control of the components involved in the reaction. However when all component are synthesized individually, there is a better control over the components concentrations, compared to producing multiple components in a cell and then purificating them. This also makes the first method more expensive.

• Membrane proteins are proteins that are embedded in the cell membrane, such as transport proteins that move molecules and ions in and out of the cell. What would be a potential challenge in expressing these proteins in live cells? What would be a potential challenge in expressing these proteins in a cell-free system?

  • The main issue to solve when to embedding a foreign protein in a live cell is for it to adapt to a complex system. So it is needed to achieve desired protein levels, so that the protein isn´t toxic, aggregate, or disrrupt the membrane stability.
  • When expressing a membrane protein in a cell free system, the correct mimicry of the cell environment is essential. The lipid composition of the synthetic membrane need to correctly mimic the one in a cell membrane, so that proteins correctly fold, and their activity isn´t affected.

• Live cells are able to constantly regenerate the necessary cellular components so they can keep producing proteins without running out of the resources they need. In a cell-free reaction, there are no live cells, so therefore, these reactions can run out of a necessary component. In order to make as much protein as long as possible, what component would you add the most of?

  • I would add as much limitant reactives as possible, meaning those reactives that are consumed during the reaction such as the energy source in form of ATP, or nucleotides. Enzymes are not consumed so they don´t need to be added in excess.

Freeze-Dried Cell Free Systems

We have learned that cell-free systems can be freeze-dried to enable the implementation of synthetic biology circuits outside of typical laboratory environments. While the demonstration of textile incorporation was reviewed here, there is wide potential for cell-free circuits to be integrated into different materials and everyday objects and devices. For this exercise, formulate a unique application for using freeze-dried cell-free circuits in a non-laboratory setting, for use in “everyday” technologies. Try to envision applications that would not normally be able to be implemented using typical biochemical reactions that require cold-chain transport and storage.

• What is your vision, and how would it be implemented?
  • I envision the integration of freeze-dried cell-free circuits into everyday household items, such as food packaging, to enable real-time monitoring of food freshness and quality. This could help diminish food-associated intoxications.
• How would this application add value in comparison to the current state of the art?
  • This application would provide consumers with an easy-to-use and cost-effective method for assessing the freshness and quality of food items without relying solely on expiration dates. It could potentially reduce food waste by alerting consumers to consume perishable items before they spoil, thereby saving money and promoting sustainability.
• Consider the constraints we talked about for freeze-dried cell-free systems: hydration, stability, operating conditions, and signal generation and detection. How will you engineer your technology to address each of these constraints?
  • Hydration: The packaging material would contain a compartment filled with a hydration solution that would rehydrate the freeze-dried cell-free circuits upon activation.
  • Stability: Specialized stabilizers would be incorporated into the freeze-dried cell-free circuits to enhance their stability and shelf life, ensuring reliable performance over an extended period. Proteins can also be engineered to achieve a maximum stabiluity
  • Operating Conditions: The cell-free circuits would be engineered to be easy to use so that common consumers are able to use them.
  • Signal Generation and Detection: The circuits would be designed to produce a visible or easily detectable signal, such as a color change, in response to specific biomarkers associated with food spoilage.
• Are there any other constraints that might be specific to your application that should be considered?
  • Safety and Regulatory Compliance: The materials and components used in the cell-free circuits must comply with food safety regulations to ensure consumer safety.