Week 9 HW: Cell-Free Systems

Homework Part A: General and Lecturer-Specific Questions

General homework questions

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 protein synthesis removes the constraints we usually face for protein synthesis when working with live cells. For example, working with live cells requires culturing cells throughout the whole cell lifecycle, with all the possibilities for error that this implies (mistakes, unexpected cell behavior due to their non-deterministic nature, etc), as well as the required timelines (speed is limited by the fundamental speed constraints from the cell growth cycle), and costs. Cell-free systems also allow for much greater control, as the main system is boiled down to its most basic functional components, removing a lot of complexity (variables outside of our control), and therefore allowing the possibility of producing much more homogeneous products.

Two special cases that CFS allow are the use of molecules that would usually be toxic for cells, and working with a much wider variety of molecules that would otherwise be destroyed by the cell, as well as modified versions of the most basic components of this machinery (that could not work in a live cell), such as amino acids that don’t exist in nature.

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

The basic components are: The lysate, which includes the “machinery”: basically ground up cells (prokaryotic or eukaryotic), providing the cellular components we need for the reactions, like ribosomes, enzymes, tRNAs and cofactors, which will translate mRNA into protein The genetic template (DNA or RNA) that encodes the product we want The “building blocks” (amino acids and nucleotides) The energy system (needed to provide and replenish the ATP and GTP needed for the reactions) (ie Mg^2+ and K+) The “environmental tuning” factors (salts, buffers, temperature, etc)

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.

Because protein synthesis is an energy-intensive process. In living cells, mitochondria handle the production of ATP, but in CFS we need to set up systems that can continually obtain the phosphates needed to convert ADP into ATP. Not being able to meet these energy needs will cause the reactions to stall or provide low yield. A method to ensure a continuous ATP supply is to include an ATP regeneration system, such as the phosphocreatine–creatine kinase system. Creatine kinase will transfer a phosphate group from phosphocreatine to ADP, regenerating ATP as it is consumed.

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

Only eukaryotic cells can perform PTM post-translational modifications (ie glycosylation), so any desired product requiring this process will require the use of eukaryotic cell lysate. This will also be required if the final desired product requires the usage of mammalian regulators. However, when these requirements are absent, prokaryotic systems are faster and cheaper.

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.

The main challenge when working with membrane proteins is that they tend to aggregate or misfold in aqueous environments (due to their hydrophobic regions trying to “escape” it)

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.

The required energy needs are not being met.

The proteins might be misfolding. In this case, we would check and tune the environmental variables, like the temperature or the salts, and possibly add chaperones or use different membrane-mimicking systems.

The final or intermediary products might be suffering degradation. In this case, I would check for the presence of nucleases, proteases, etc.

Homework Question from Kate Adamala:

Pick a function and describe it. What would your synthetic cell do? What is the input and what is the output?

My synthetic cell would be built to capture environmental CO2. The minimal inputs would be an energy source for carbon fixation (ATP), CO2, inorganic nutrients (phosphates, salts, enzymatic cofactors), and a carbon concentration mechanism. I would also need a metabolic mechanism for carbon fixation, such as a rubisco-based Calvin cycle.The output would be a stable storage mechanism in the form of carbonates, to safely lock away carbon in the system.

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

Probably not, as carbon fixation and compartmentalization would require additional metabolic processes beyond basic protein translation.

Could this function be realized by genetically modified natural cell?

Yes, and pretty easily as there are already many organisms that perform these functions.

Describe the desired outcome of your synthetic cell operation.

I would aim to make this operation as stripped down as possible, trying to create the minimal viable carbon capturing mechanism from a cell-free system. The desired outcome should be a minimal measurable function of carbon processing and storage, the cell being “alive” is not needed. I would aim for the synthetic cell to continuously convert dissolved CO2 into measurable reduced carbon for several hours while maintaining internal biochemical activity.

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

What would be the membrane made of?

I’d want a stable membrane permeable to CO2, such as a lipid membrane inspired by bacteria, composed of mostly POPC and POPE.

What would you encapsulate inside? Enzymes, small molecules.

I would need plasmids encoding DNA and tRNAs + ribosomes for translation, PEP and pyruvate for ATP regeneration, salts and cofactors such as Mg, phosphate, potassium etc.

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 system will suffice.

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

Membrane pores for passive diffusion will work well for small molecules, membrane channels to retain larger proteins (Outer Membrane Protein F)

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.)

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cholesterol (for membrane), cardiolipin Genes: rbcL, rbsS (rubisco), prk, csoSCA, ompF, pykF

How will you measure the function of your system?

HPLC assays can provide product quantification to show if CO2 has been converted into reduced carbon in the synthetic 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:

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

Portable pathogen sensor patches embedded in public transport vehicles to monitor the load of airborne pathogens How will the idea work, in more detail? Write 3-4 sentences or more.

By using a freeze-dried detection system coupled with an RNA switch or CRISPR-based sensor, this device could be carried by rideshare or transport service providers to periodically monitor pathogen levels inside a vehicle using a simple swab test. The system could potentially be integrated into seatbelts, door panels, or other interior surfaces as a small patch or sticker.

What societal challenge or market need will this address?

This technology could help identify when a vehicle requires more thorough disinfection, improving both driver and passenger safety. For example, immunocompromised riders could potentially request vehicles with lower measured pathogen loads. In addition, the system could provide useful public health data for applications such as outbreak monitoring, epidemic detection, and prevention.

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

The device itself could take the form of a disposable patch containing a cartridge with the required water and reaction buffers, activated through a simple button or switch that mixes the components (such as at the end of a driver’s shift). A single-use format replaced daily would help minimize contamination risks, while the freeze-dried design could maintain stability for approximately 3–6 months at room temperature.

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!

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/ .

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)

Future human missions to Mars will require sustainable systems for food production, waste recycling, and oxygen generation. However, Mars presents extreme conditions, including high radiation, low temperatures, and limited nutrients, making microbial survival difficult. My proposal investigates how bacterial stress-resistance pathways could be engineered and tested using the BioBits cell-free protein expression system to identify genes that improve survival under Mars-like conditions. Understanding how to design hardier microbes is significant for supporting long-term space habitation, scientifically interesting for studying adaptation to extreme environments, and relevant to developing biotechnology for extraterrestrial living.

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)

Genes and proteins involved in bacterial salt tolerance, including osmotic stress-response pathways, to understand survival under high-salinity Mars-like conditions.

Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) Mars soil is believed to contain high concentrations of salts, which can damage cells by causing dehydration and disrupting biological processes. By studying bacterial salt-tolerance genes and proteins, my project aims to identify mechanisms that help microbes survive in high-salinity environments. Using the BioBits cell-free system, we can test stress-response pathways under simulated Mars-like salt conditions to better understand how bacteria might be engineered to survive and support future human missions through waste recycling, food production, or oxygen generation.

Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) The goal of this project is to determine whether bacterial genes associated with salt resistance can help maintain biological activity in environments with high salinity, similar to conditions expected on Mars. I predict that introducing these stress-response genes into the cell-free system will result in more effective protein production under salty conditions compared to systems without them. This idea is based on the observation that some Earth bacteria naturally survive in highly saline habitats by activating protective mechanisms against osmotic stress. Identifying genes that remain functional in harsh environments could contribute to developing microbes better suited for supporting long-term human space exploration.

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)

This experiment will use the BioBits cell-free protein expression system to test bacterial salt-tolerance genes under increasing salt concentrations that mimic Mars-like conditions. Samples will include reactions containing salt-resistance genes and control reactions without these genes. Multiple salinity levels will be tested to compare protein expression across environments. Protein production will be measured using fluorescent reporters and visualized with a P51 Molecular Fluorescence Viewer. Data collected will include fluorescence intensity under different salt conditions to determine whether salt-tolerance genes improve biological activity in high-salinity environments.