Week 9 HW: Week 9 — 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.

Cell free expression allows teams to conduct biomanufacturing without living cells and operate beyond the constraints of productions in living systems.

Two cases where cell-free expression is more beneficial than cell production are where:

A) biomanufacturing would kill the cells B) teams have a desire to rapidly prototype biomanufacturing workflows after computational modeling

  1. Describe the main components of a cell-free expression system and explain the role of each component.

The main components of cell free expression are as follows:

Cell lysate/mix: These provide the vital translation components, in addiiton to ribosomes, other enzymbes, and tRNAs Genomic Template: These are needed to encode and develop the protein of interest Salts: Ioinic condition maintenance Buffer: Maintaining pH Cofactors/additives: Promoting enzymatic activity Amino Acids: These supply building blocks for translation Energy System: These supply energy to power transcription and translation

  1. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

Energy provision generation is critical to sustain reactions. Supplying molecular energy packs that can regenerate ATP during a reaction can assist this.

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

Eukaryotic and prokaryotic cell-free expression systems have their own unqiue advanges. Prokaryotic cell-free expression tends to be great for rapid prototyping and is robust. Eukaryotic systems can have advantages with complex products by which post-translational modification may be desired.

I’d possibly consider developing flourescent proteins in prokaryotic systems while focusing on antibodies with eukarytic systems.

  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.

A) My initial guess would be to examine setups by which I can utilize detergents and or membrane disaggrgating components and trial setups. B) Challenges come from aggregation of membrane proteins, insolubility of components, and reduced yields. I would possibly consider different spatial component arrangements, release modalities, temperature changes, and experiment with amounts of disaggrgating components.

  1. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.

Three possible reasons could be a poor template, poor environmental considtions, and or lack of energy. Troubleshooting respectively would look like: attempting template optimization, environmental optimization, and trialing suppying more energy sources for the reaction. Each of these would be systematically trialed.

Homework question from Kate Adamala

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

  1. Pick a function and describe it.

One function is fluorescence in the presence of a target molecules.

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

This cell would fluoresce with response to environmental exposure to pesticides. The input is the pesticide and the output is fluorescence.

    1. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

This could be.

    1. Could this function be realized by genetically modified natural cell?

Yes.

    1. Describe the desired outcome of your synthetic cell operation.

The desired outcome would be a deployable cell that could be used to find and determine pesticide levels.

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

The needed components would be an array of cell-regulatory components, amino acides, co-factors, salts, an energy regeneration system, NTPs, amino acids, a DNA template, a DNA template encoding reporter, and cell-free transcription and translation machinery all within a liposome.

    1. What would be the membrane made of?

The membrane could be made of a phospolipid lipsome

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

Everything that needs to be encapsulated would be. Enzumes and small molecules alone is insufficient.

    1. 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 promotors, like Tet-ON, you need mammalian)

A bacterial sourrce, like E. Coli could suffice.

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

The synthetic cell could communicate with the environment through membrane bidirectional membrane exhange of elements, followed by fluorescence in the preosence of the pesticides. What is assumed is that the pesticides pass through the membrane and can interact with the cellular internal machinery.

  1. Experimental details

To Be Added

    1. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)

Cholesterol (lipid)

eqFP611 (Gene that codes for Electra2)) See: https://www.fpbase.org/protein/electra2/

    1. How will you measure the function of your system?

I could measure the flourescene with a plate reader, compatible microscope, and or similiar 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:

To Be Added

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

This pitch is for a freeze-dried cell-free biosensor that turns blue in the presence of caffeine to help caffeine-sensitive consumers identify highly caffeinated drinks.

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

The system would contain a freeze-dried cell-free transcription/translation mixture and a caffeine-responsive genetic circuit. When added to a beverage, caffeine would activate the sensing system and induce expression of a visible reporter protein such as Electra2 or a colorimetric enzyme output. The reaction would produce a detectable color change within a short time period, allowing users to estimate whether caffeine is present in the drink.

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

This system could help caffeine-sensitive individuals avoid accidental caffeine exposure in beverages where caffeine content is poorly labeled, inconsistent, or unknown. It may also support rapid beverage testing in restaurants, cafes, or consumer safety settings.

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

Stability could be improved through lyophilization additives such as trehalose and by storing the reaction mixture in sealed moisture-resistant packaging until use. The system is likely single-use after hydration, so the biosensor could be designed as a disposable low-cost test strip or reagent capsule. Past that, I could address the limitation by simply listing warnings for ingredients that could complicate the reaction process.

Homework question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

To Be Added

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/ .

To Be Added

  1. Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words)

Pivoting the prior example:

Astronauts experience sleep disruption and fatigue during long-duration space missions. Lightweight biochemical sensors could help monitor stimulant exposure in environments with limited laboratory equipment. Freeze-dried cell-free systems are promising because they are portable, stable, and programmable.

  1. Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words)

A caffeine-responsive genetic circuit (core possibly being the CYP1A2 gene) linked to a fluorescent or color-changing reporter protein in a freeze-dried cell-free system.

  1. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

A caffeine biosensor could help astronauts monitor stimulant exposure during missions where sleep and alertness are important. Freeze-dried cell-free systems are useful because they are compact, shelf-stable, and easy to activate with water.

  1. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

I hypothesize that a freeze-dried cell-free biosensor can detect caffeine and produce a measurable fluorescent or colorimetric signal after hydration. The goal is to test whether reporter intensity changes with caffeine concentration and whether the system remains stable after storage.

  1. Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

Freeze-dried cell-free reactions would be rehydrated with different caffeine concentrations, under a variety of common environmental conditions normal to the astronauts. Negative controls would contain no caffeine. Reporter output would be measured using fluorescence or visible color change with a flourescence reader. Stability after storage would also be tested.

Homework Part B:

Put your chosen final project slide in the appropriate slide deck following the instructions on slide 1: MIT/Harvard/Wellesley ONE FINAL PROJECT IDEA Committed Listener ONE FINAL PROJECT IDEA

Done.

Submit this Final Project selection form if you have not already.

Done.

Begin planning how you will write your final project documentation based on these guidelines

Done.

Prepare your first DNA order and put it in the “Twist (MIT)” or “Twist (Nodes)” tab of the 2026 HTGAA Ordering: DNA, Reagents, Consumables spreadsheet, as appropriate.

Coordinated with Node. Not having lab access, I was excused from needing to order a physical construct

First Twist order deadline for MIT/Harvard/Wellesley students is Friday, April 3 at 11PM ET First Twist order deadline for Committed Listeners is Friday, April 10 at 11PM ET. (Your Node Lead will place the Twist order, so please work with them to finalize your constructs and ordering decisions.)

Coordinated with Node. Not having lab access, I was exempt from needing to order a physical construct.