Week 9 HW: Cell Free Systems

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.

CFPS offers several key advantagesin felxibility and experimental control over traditional in vivo protein expression. In terms on flexibility, unlike living cells, we have an open reaction enevironment as we are not constrained by structures or the viability of the cell. We can add and remore components at any time. As we dont depend on the cell, there is no need for cloning or transformation, allowing the qucik testing of multiple gene constructs at the same time. We can express proteins that coudl be toxic or unstable in livign things because nothing is alive. Comparing the benefits for control of the experimental values, CFPS systems are superior as we cna tightly control conditions, directly manipulate the gene expression and define the environment for production.

Some situations where CFPS is more beneficial than the cell based production:

  • studying and needing to produce toxic proteins that could harm the host.

  • incorporating non standard aminoacids, as there are no metabolic constrains.

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

Components:

  • DNA/mRNA template: is the manual for the encoding of the target protein. DNA is trancribed to mRNA and then translated into the protein.

  • Energy supply (ATP/GTP): we need energy for the transcription, translation and protein folding steps.

  • Aminoacids: they are the building blocks of the protein.

  • Nucleotides: they are required for the trancription of DNA.

  • Cofactors + salts: mantains enzyme activity and ribosome stability.

  • Cell extract: the needed molecular machinery (ribosomes/tRNAs/aminoacyl-tRNA synthase/initiation-elongation factors) for transcription and translation.

  • 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.

The process of protein sysnthesis is energetic demanding because of the need of 4ATP molecules for peptide bond, without any regeneration ATP is finished quickly and by products accumulate and inhibit reactions.

A method we could use to ensure the continuous ATP supply could be a PEP system, as it acts as a high energy phosphate donor to regenerate the ATP. It is a pretty simple method and has a 1:1 rate.

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

Prokaryotic CFPSs are fast and low cost but are pretty limited when it comes to protein folding and post translational modifications. Eukaryotics are slower and high cost but have a better result for the protein folding and have a variability of available ways for post translational modifications.

So if I had to choose proteins for each to synthetize according to their characteristics; I would produce GFP in a prokaryote system as it is a simple protein that doesnt need a high level folding or any modifications. For the eukaryote system, a good protein to produce could be any fragment of an antibody, as it makes it possible to do proper folding and post transcriptional mdofications.

  • 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 design a membrane protein expression some of the challanges that we could see if the is the aggregation of the proteins, the misfolding of them outside the membrane environment and the chance of them having a low solubility. We could adress some of this challanges starting by optimizing the conditions for the protein sysnthesis so there are no holdbacks on the sysnthesis. For the problems with folding we could assist the process with the add on of chaperones for assitance in insertion and folding. For the solubitlity and overall it troubleshooting as it not being in the membrane environment, we could try to recreate this ecosystem by addign membrane mimetics and give a lipidic enviroment for the translation.

  • 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.

  • Protein Aggregation: we could solve it by adding chaperonse and solubility tags to the system.

  • Energy Depletion: we could change to a sustainable energy system so there’s no chance for having an insufficient amount of ATP.

  • Transcription Problems: if we have a weak promoter or degraded dna we have problems with the trancription step. We can fix it by changing the promoter or changing the dna/rna template if it’s damaged.

Homework question from Kate Adamala

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

  1. Pick a function and describe it.

A synthetic minimal cell with the fucntion of detecting and staging liver damge by sensing a panel of biomarkers representing early injury, hepatocyte damage and cell death.

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

the cell would detact the input of serum/blood and detect the biomarkers (miR-122, ALT, AST, GLDH, CK18, OPN, ammonia, lactate) and give an output of flourescence patterns according the stage of damage.

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

In theory most of it can function without it but the encapsulation allows the modular and independent sensing of each biomarker.

  • Could this function be realized by genetically modified natural cell?

Yes, a modififed cell could perform similar sensing,but a synthethic cell is safer and more controllable avoiding any livign cell complications as we are looking at some biomarkers that could damage the cell.

  • Describe the desired outcome of your synthetic cell operation.

The desire outcome is a stage diagnostic output so we coudl distinguish early liver injury from severe liver failure without the need of invasive procedures.

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

Biomarker mir122: Promoter 17 + (creation of toeswitch seq complementary to miR-122) + RBS + GFP (reporter) + Terminator T7.

The biomarker binds to the toeswitch and open rna hairpin allowing the reporting signal to come thru.

Biomarker gldh / ck18 : this protein needs to be detected via aptamer/antibody giving a dna tirgger to activate the signal.

  • What would be the membrane made of?

The membrane could be made of phospholipid vesicles with cholesterol and the addition of membrane pores for protein entry to facilitate the uptake of certain biomarkers.

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

Inside the membrane the encapsulated contents would be the cell free trancription/translation system and the dna constructs for each biomarkers.

  • 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)

We could use bacterial (e.coli) system as it is sufficient for the biosensor. Mammalian system are not needed as we dont need to modulated promotors, just need the genetic circuits and the protein reactions with the defined biomarkers.

  • 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 communication with the enviroment comes from the free diffusion of the small molecules part of the biomarkers and the big proteins needed to be sense envir via channels in the membrane or can be converted to dna triggers.

  1. 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.)
  • POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) – forms the main phospholipid bilayer
  • Cholesterol – stabilizes the membrane and controls fluidity
  • OmpF (gene: ompF, UniProt P02931) – membrane protein channel to allow protein or metabolite entry
  • miR-122: T7 promoter + miR-122 toehold switch + RBS + GFP + T7 terminator (Blue fluorescence)
  • GLDH: Protein-binding aptamer → DNA trigger → T7 promoter → YFP + T7 terminator (Yellow fluorescence)
  • CK18: Protein-binding aptamer → DNA trigger → T7 promoter → YFP + T7 terminator (Yellow fluorescence)
  • How will you measure the function of your system?

The function of the system is gonna be measured by flousrescence from each reporter. The combination of the colors are gonna be used to understand which stage we are looking at.

Bibliography

  • Church, R. J., Kullak-Ublick, G. A., Aubrecht, J., Bonkovsky, H. L., Chalasani, N., Fontana, R. J., Goepfert, J. C., Hackman, F., King, N. M. P., Kirby, S., Kirby, P., Marcinak, J., Ormarsdottir, S., Schomaker, S. J., Schuppe-Koistinen, I., Wolenski, F., Arber, N., Merz, M., Sauer, J. M., … Watkins, P. B. (2019). Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collaborative effort. Hepatology, 69(2), 760–773. https://doi.org/10.1002/hep.29802

  • He, F., Wang, Q., Li, J., & Ma, X. (2023). The application of aptamer in biomarker discovery. Biomarker Research, 11(1), Article 26. https://doi.org/10.1186/s40364-023-00510-8

  • Kandemir, H., Cinar, Y., & Ozturk, M. (2024). Serum microRNA-122 for assessment of acute liver injury in patients with extensive skeletal muscle damage. Laboratory Medicine, 55(5), 585–591. https://doi.org/10.1093/labmed/lmae022

  • Vliegenthart, A. D., Berends, J. E., Mashimo, T., Wouters, E. P. A., Verheij, J., & Stoopen, G. M. (2018). A longitudinal assessment of miR-122 and GLDH as biomarkers of drug-induced liver injury in the rat. Biomarkers, 23(4), 303–312. https://doi.org/10.1186/s40364-023-00510-8

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.

An intelligent bio reactive textile that uses lyophilized cell free sensors to monitor real time thermal stress and autonomously tiggers cooling via a enzyme resposonse.

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

The system would be done with a layered bio circuit consisting on a biosensor embedded in textile. One part is the rna thermomether that expresses a color changing protein to provide the visual map. While the second is an enzymatic trigger that produces a protease enzyme when the threshold temperature is reach, digests microcapsules and releases a cooling agent, making the temperature drop and cooling the person.

  • What societal challenge or market need will this address?

There are currently cooling vests but there are no direct cooling systems textiles that sense when they are needed. The idea was born with the f1 in mind but can be stablisihed in any environment where high stress and physical exhaustion from heat is present. The system provides a failsafe for the body to mantain its cooling in extreme conditions.

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

For the need to its activation with water, divert to the use of the sweat as a biological switch on. The lyophilized system can be engineered to turn on when only the correct salt/pH is given from the human perspiration.

One-time used limits a lot of cfs ideas but for this project we could create replacible biopatches. Instead of throwing away a whole uniform, patches could be taken out and replaced when needed lowering the waste.

Bibliography

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)

Long duration spaceflight is associated with hemolytic anemia caused by the increased red blood cell destruction. Factors like microgravity membrane changes, fluid shifts and oxidative stress form space radiation contribute to this anomaly. Understanding this mechanisms are essential to astronauts health as it presents itself during extended mission and continues for a long period after being back on Earth. Cell fre systems provide a simplified platform to investigate protein stability under these oxidative conditions can model blood cell damage helping us develop protectice strategies.

  • 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)

GFP as a reporter for protein stability in bacteria cell free systems. Optionally red human blood cells proteins for a conceptual mammalian system.

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

Oxidative stress contirbutes to the red blood cell damage in astronauts, and with the BioBits system, gfp flourescence reflects protein integrety under stress modelling this conditions and its affects in RBCS. Conceptually a mammalian system expressing hemoglobin or cytoskeletal proteins could be a more direct human model to rbc vulnerability.

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

This project hypothesis is that oxidative stress conditions reduce portein stability and function decreasing the flourescence in the cell free system. With the biobits experiment, gfp reports the model protein damage, demostrating the effects of the condition can impair the protein integrity. These changes would indicate protein misfolding or degradation, analogous to damage occurring in astronaut red blood cells. Ultimately, this work provides insight into mechanisms underlying hemolytic anemia and demonstrates how biotechnology can support astronaut health on long-duration missions.

  • 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)

e experiment will use freeze-dried BioBits cell-free reactions expressing GFP. Samples will include: (1) control reactions under normal conditions, and (2) reactions exposed to oxidative stress (e.g., hydrogen peroxide). Fluorescence intensity will be measured using the P51 Molecular Fluorescence Viewer to assess protein stability. Conceptually, a mammalian cell-free system expressing human RBC proteins (hemoglobin or spectrin) could be similarly tested under oxidative stress or simulated microgravity, with fluorescence monitoring protein integrity. Comparing stressed and control samples allows evaluation of stress-induced protein damage, modeling mechanisms contributing to hemolytic anemia in astronauts.

Bibliography