Week 9 HW: Cell - free systems

Homework Part A: General and Lecturer-Specific Questions

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 protein synthesis provides greater flexibility and control compared to traditional in vivo expression, as the reaction occurs outside living cells, allowing factors such as DNA concentration and energy components to be adjusted without affecting cell viability. In in vivo expression methods, amplifying the bacterial plasmid, sequencing it, expressing the strain, and purifying the protein must be done separately for every single protein in the pathway. In contrast, in cell-free protein expression using a synthetic cell, proteins can be expressed directly from linear PCR fragments without the need for plasmid construction. This makes the process much more time‑efficient and allows results to be obtained within hours. Furthermore, all proteins can be expressed in one pot by adjusting the concentrations of the DNA, unlike in vivo systems that require different promoters for each protein and expression level.

Answer based on Kate Adamala, 2/03/2026 lecture

Cases where cell-free expression is more beneficial than cell production:

1. In the production of toxic or membrane proteins, cell-free systems are advantageous because the reaction occurs outside a living cell. Internal metabolism does not need to be maintained, and there are no cellular barriers limiting translation control (Zemella et al., 2015). Traditionally, genetic circuit prototyping using plasmid DNA is time‑consuming, taking several days per validation cycle. In contrast, linear DNA can complete the same cycles within 4–8 hours, making it possible to validate large circuits rapidly and to study molecules that were previously considered too toxic for in vivo work (Brookwell et al, 2021).

2. Cell-free systems also benefit other prototyping efforts, such as drug discovery, by shortening the time from compound identification to validation. Moreover, reactions can be monitored in real time, providing insights into the underlying mechanisms and improving the design and optimisation of experimental pathways.

References

  • Brookwell, A., Oza, J.P. and Caschera, F. (2021). Biotechnology Applications of Cell-Free Expression Systems.Life , 11(12), p.1367. doi:https://doi.org/10.3390/life11121367.
  • Zemella, A., Thoring, L., Hoffmeister, C. and Kubick, S. (2015). Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. ChemBioChem, [online] 16(17), pp.2420–2431. doi:https://doi.org/10.1002/cbic.201500340.

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

    A cell-free expression system contains all the molecular machinery needed for transcription and translation without using living cells. Its main components include:

  • Lipid membrane: Composed of phospholipids for structural stability and cholesterol additives that increase membrane fluidity and durability.
  • Cell extract: Provides ribosomes, enzymes, and other necessary cofactors for transcription and translation.
  • Cytoplasmic components: Small molecules such as salts, metabolites, and energy substrates that support biochemical reactions.
  • tRNAs and nucleotides: Supply the genetic code and building blocks for protein synthesis.
  • Membrane channels: Allow controlled exchange of small molecules, enabling communication and regulation.
  • Genetic template: A minimal genome in the form of a plasmid or linear DNA containing the genes to be expressed.

    • Answer based on Kate Adamala, 2/03/2026 lecture

    3. 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 regeneration is critical in cell-free systems because protein synthesis requires a continuous supply of ATP. Translation is highly energy‑intensive, using roughly 4–5 ATP equivalents per peptide bond. As the reaction proceeds, phosphate by‑products accumulate and deplete the energy pool, limiting protein yield. To maintain ATP levels, energy regeneration schemes can be introduced.

    One effective example is the Protein Synthesis Using Recombinant Elements (PURE) system, a minimal biochemical setup capable of carrying out cell‑free protein synthesis using defined enzymatic components. In this system, pyruvate oxidase is added to catalyse the conversion of pyruvate and inorganic phosphate into acetyl phosphate. This intermediate can then regenerate ATP through its conversion to acetate, catalysed by acetate kinase naturally present in the extract(Yadav et al., 2025).

    references

  • Yadav, S., Perkins, A.J.P., Liyanagedera, S.B.W., Bougas, A. and Laohakunakorn, N. (2025). ATP Regeneration from Pyruvate in the PURE System. ACS Synthetic Biology, 14(1), pp.247–256. doi:https://doi.org/10.1021/acssynbio.4c00697.

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

    Prokaryotic cell-free systems typically use E. coli extracts and provide high protein yields at low cost. They are rapid to prepare and are easily genetically engineered, which makes them suitable for large-scale production and the incorporation of non-canonical amino acids. However, they have limited capacity for complex protein folding and lack most post-translational modifications. In contrast, eukaryotic cell-free systems use extracts from sources such as wheat germ, insect cells, or protozoa. They are better suited for proteins that require proper folding and post-translational modifications, allowing the production of more complex and functional proteins. However, they are generally slower, more expensive, and often yield less protein than prokaryotic systems (Zemella et al., 2015).

    For eukaryotic systems: Monoclonal Antibodies (mAbs)

    These are complex, large glycoproteins that require proper folding and post-translational modifications, which eukaryotic systems handle better. Heavily used in drug manufacturing, their higher solubility in eukaryotic extracts makes the final drug more effective (Ding and Huang, 2024).

    For prokaryotic systems: Human Serum Albumin (HSA)

    This small protein needs no complex glycosylation. Commonly used as a drug carrier where production time matters, prokaryotic systems provide the high yields needed for scalable manufacturing (Raoufinia et al., 2016).

    references

  • Creative Biostructure (2025). Protein Synthesis in Prokaryotes vs. Eukaryotes: What’s the Difference? [online] Creative-biostructure.com. Available at: https://www.creative-biostructure.com/protein-synthesis-in-prokaryotes-vs-eukaryotes.htm [Accessed 4 Apr. 2026].
  • Ding, Z. and Huang, Y. (2024). Production of Monoclonal Antibodies for Therapeutic Purposes: Applications, Techniques, and Improvement. doi:https://doi.org/10.20944/preprints202405.1854.v1.
  • Zemella, A., Thoring, L., Hoffmeister, C. and Kubick, S. (2015). Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. ChemBioChem, [online] 16(17), pp.2420–2431. doi:https://doi.org/10.1002/cbic.201500340.
  • Raoufinia, R., Mota, A., Keyhanvar, N., Safari, F., Shamekhi, S. and Abdolalizadeh, J. (2016). Overview of Albumin and Its Purification Methods. Advanced Pharmaceutical Bulletin, 6(4), pp.495–507. doi:https://doi.org/10.15171/apb.2016.063.

    • 5. How would you design a cell-free experiment to optimise the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

    What needs to be added:

  • Insoluble expression:If lipids or detergents are not included, membrane proteins tend to aggregate due to their hydrophobic regions. Although aggregation can sometimes allow refolding into functional proteins, it more often results in misfolded or inactive proteins.
  • Detergents: These are commonly added during expression to increase solubility by surrounding hydrophobic regions of the protein. However, they can sometimes destabilise the protein or interfere with downstream applications.
  • MSP-Nanodiscs:These provide a stabilising lipid environment for membrane proteins. They enable proteins to remain in a near-native state and can be used in a wide range of assays without requiring detergents.
  • Liposomes and lipids:These create a membrane-like environment that supports proper folding and insertion of membrane proteins, particularly when used with insect cell lysates.
  • There are two main methods of cell-free membrane protein expression: batch and Continuous exchange cell-free (CECF) / dialysis systems. I would choose CECF/dialysis methods over batch systems because they allow longer reaction times and generally result in higher protein yields.

    • (Cube Biotech, 2014)

    Steps:

    Follow a medium-scale protein production approach, which results in a high protein yield whilst optimising reaction conditions for maximum purity:

  • Preparation of plasmid template: Use circular DNA templates, as they are more stable and optimised for high-yield protein expression.
  • Reaction setup: Use a large reaction volume within a continuous exchange system to support sustained protein synthesis.
  • Reaction conditions optimisation: Maximise yield by adjusting key variables such as the composition of the feeding solution.
  • Protein analysis: Assess the yield and purity of the expressed protein and prepare it for downstream applications.

    • (Synthego, 2026)

    Challenges and how to address them:

    Membrane proteins are difficult to study because their hydrophobic surfaces require detergents or lipid environments for extraction and stabilisation. This creates challenges not only during expression, but also throughout purification and downstream analysis.

    In addition, membrane proteins are often flexible and structurally unstable, which reduces the likelihood of obtaining a functional or well-behaved protein. This issue can be addressed by providing a more native-like environment during expression, such as incorporating lipids or nanodiscs to improve stability and folding. These inherent properties impact multiple stages of research, including expression, solubilisation, purification, crystallisation, and structure determination. As a result, it is often necessary to adopt broader screening strategies.

    One way to overcome these challenges is to test a range of targets or homologues, increasing the chances of identifying a protein that behaves well under experimental conditions. This approach has been particularly successful in the study of G-protein-coupled receptors, where reducing protein flexibility has been essential for structural studies. For example, stabilising flexible regions through engineered modifications or binding partners has enabled successful structure determination.

    (Carpenter et al., 2008).

    references

  • Carpenter, E.P., Beis, K., Cameron, A.D. and Iwata, S. (2008). Overcoming the challenges of membrane protein crystallography. Current Opinion in Structural Biology, 18(5), pp.581–586. doi:https://doi.org/10.1016/j.sbi.2008.07.001.
  • Cube Biotech (2014). Why use cell-free protein expression? [online] Cube Biotech. Available at: https://cube-biotech.com/our-science/cell-free-lysates/cell-free-expression/ [Accessed 4 Apr. 2026].
  • Synthego (2026). Cell-Free Protein Expression: A Guide - Synthego. [online] Synthego. Available at: https://www.synthego.com/cell-free-protein-expression-guide/ [Accessed 4 Apr. 2026].

    • 6.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.

    Reasons for low yield of target protein:

    1)Low ATP Level: Use Protein Synthesis Using Recombinant Elements (PURE) for ATP regeneration. (Yadav et al., 2025) Poor DNA template: Use circular DNA to increase stability. (Synthego, 2026)

    Poor protein folding: Add protein folding factors such as Chaperones to the in vitro extract. (Anantha, 2025)

    references

  • Anantha, S. (2025). Solved: Low Yields in Cell-Free Protein Synthesis. [online] Bitesize Bio. Available at: https://bitesizebio.com/10234/solvedlow-yields-in-cell-free-protein-synthesis/ [Accessed 4 Apr. 2026].
  • Synthego (2026). Cell-Free Protein Expression: A Guide - Synthego. [online] Synthego. Available at: https://www.synthego.com/cell-free-protein-expression-guide/ [Accessed 4 Apr. 2026].
  • Yadav, S., Perkins, A.J.P., Liyanagedera, S.B.W., Bougas, A. and Laohakunakorn, N. (2025). ATP Regeneration from Pyruvate in the PURE System. ACS Synthetic Biology, 14(1), pp.247–256. doi:https://doi.org/10.1021/acssynbio.4c00697.

    • Homework question from Kate Adamala

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

    Pick a function and describe it.

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

    Based on my Final Project proposal: A synthetic minimal cell for PAH paint bioremediation.

  • Function: Continuously degrades PAH carcinogenic pollutants produced by incomplete combustion (eg. traffic emissions and industrial processes) when deposited on the paint surfaces.
  • Input: Gaseous PAHs from air pollution
  • Output: Carbon dioxide and water —> PAH-free surfaces

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

    No, this function could not be realised by cell-free Tx/Tl alone and would require encapsulation. Encapsulation would protect the synthetic minimal cells within the paint from harsh outdoor environmental conditions, allowing the PAH-degrading enzymes to function effectively.

    c) Could this function be realized by genetically modified natural cell?

    A genetically natural cell could realise PAH degradation, but would face serious challenges. PAHs like benzo[a]pyrene (BaP) form toxic reactive diol epoxides (BPDE) that bind to DNA adducts that halt replication and enzyme production (Wang et al., 2023). Additionally, living cells also need complex nutrients for long-term survival, plus growth control to prevent overgrowth.

    However, in my project proposal examining B. subtilis's PAH-degrading properties, its spore-forming ability and environmental robustness. In relation to food source, a study conducted showed strain BMT4i (MTCC 9447) utilising BaP as a sole carbon and energy source with 84.66% degradation efficiency (Lily et al, 2010). Additionally, the degradation capacity of microbes may be induced by exposing them to higher PAH concentrations, resulting in genetic adaptation or changes responsible for high-efficiency removal/degradation. Overall, this keeps it as a strong candidate for the development of a PAH-degrading paint (Sakshi and Haritash, 2020).

    references

  • Hazan, A., Lee, H.Y., Tiong, V. and AbuBakar, S. (2025). Bacillus subtilis Spores as a Vaccine Delivery Platform: A Tool for Resilient Health Defense in Low- and Middle-Income Countries. Vaccines, [online] 13(10), pp.995–995. doi:https://doi.org/10.3390/vaccines13100995.
  • Lily, M.K., Ashutosh Bahuguna, Koushalya Dangwal and Garg, V. (2010). Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447). Annals of Microbiology, 60(1), pp.51–58. doi:https://doi.org/10.1007/s13213-009-0010-y.
  • Sakshi and Haritash, A.K. (2020). A comprehensive review of metabolic and genomic aspects of PAH-degradation. Archives of Microbiology, 202(8), pp.2033–2058. doi:https://doi.org/10.1007/s00203-020-01929-5.
  • Wang, H., Liu, B., Cui, H., Xu, P., Xue, H. and Yuan, J. (2023). Dynamic changes of DNA methylation induced by benzo(a)pyrene in cancer. Genes and Environment, 45(1). doi:https://doi.org/10.1186/s41021-023-00278-1.

    • d) Describe the desired outcome of your synthetic cell operation.

    PAH-contaminated paint surfaces become PAH-free, preventing re-volatilisation and runoffs.

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

    a) What would be the membrane made of?

    Phospholipid bilayer with additive cholesterol (increases stability and fluidity) for paint surface adhesion and environmental stability. Additionally, PEG-lipids (polyethene glycol-lipids) as it acts as an interface between hydrophobic polymer cores and aqueous environments (Esteve, 2025).

    References:

  • Esteve, M.Á. (2025). Beyond Lipids and Polymers: Understanding Hybrid Nanoparticles. [online] Curapath.com. Available at: https://blog.curapath.com/beyond-lipids-and-polymers-understanding-hybrid-nanoparticles [Accessed 4 Apr. 2026].

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

  • Catabolic enzymes such as dehydrogenase, monooxygenase, catechol dioxygenase, and aromatic-ring-hydroxylating dioxygenase (found in bacterias gene that degrade PAHs, key components of the pathway). (Tesfaye et al., 2025)
  • Linear DNA encoding for the catabolic enzymes.
  • E. Coli cell extract containing tRNAs, ribosomes and polymerases.
  • Cholesterol and phospholipids.

    • references

  • Tesfaye, E.L., Bogale, F.M. and Aragaw, T.A. (2025). Biodegradation of polycyclic aromatic hydrocarbons: The role of ligninolytic enzymes and advances of biosensors for in-situ monitoring. Emerging Contaminants, [online] 11(1), p.100424. doi:https://doi.org/10.1016/j.emcon.2024.100424.

    • c) 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)

    I would use E. Coli as my Tx/TI bacterial system, as PAH degradation is present in prokaryotic enzymes and pathways; therefore, no mammalian systems would be needed. Additionally, they also give a higher yield production and have a lower cost compared to eukaryotic and mammalian systems.

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

    The catabolic PAH-degrading enzymes are hydrophilic proteins that remain inside the cell extract. The PAHs (small, hydrophobic) are what will passively diffuse through the phospholipid membrane and due to their high permeability, no specific channels need to be expressed (Cao et al., 2021).

    references

  • Cao, Y., Zhang, L., Geng, Y., Li, Y., Zhao, Q., Huang, J., Ning, P. and Tian, S. (2021). Evaluation of the permeability and potential toxicity of polycyclic aromatic hydrocarbons to pulmonary surfactant membrane by the parallel artificial membrane permeability assay model. Chemosphere, p.132485. doi:https://doi.org/10.1016/j.chemosphere.2021.132485.

    • 3. Experimental details

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

    Lipids:

  • Cholesterol
  • Polymer coating

    • Specific genes:

  • For ATP regeneration: pyruvate oxidase and acetate kinase
  • Catabolitic PAH-degrading enzymes: dehydrogenase, monooxygenase, catechol dioxygenase, and aromatic-ring-hydroxylating dioxygenase

    • b) How will you measure the function of your system?

    Use Mass spectrometry coupled to gas chromatography (GC-MS) quantification to determine the exact levels of volatile compounds (VOCs), specifically the PAHs (Dimitrios,2024), present before and after application of paint.

    references

  • Dimitrios Tsikas (2024). Perspectives of Quantitative GC-MS, LC-MS, and ICP-MS in the Clinical Medicine Science—The Role of Analytical Chemistry. Journal of Clinical Medicine, [online] 13(23), pp.7276–7276. doi:https://doi.org/10.3390/jcm13237276.

    • 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.

    I will continue to apply my final project proposal from above to explore my development options.

    Self-monitoring, self-remediating anti-pollution paint that continuously degrades volatile Polycyclic Aromatic Hydrocarbons (PAH) using freeze-dried Bacillus subtilis BMT4i expressing PAH-degrading enzymes.

    What societal challenge or market need will this address?

    PAHs are associated with urban pollution sources such as vehicle exhaust and industrial emissions, which persist in the air and soil and pose significant health risks via inhalation, ingestion, and dermal contact (Wu et al., 2025). This freeze-dried cell-free system addresses these issues and could be a promising tool for environmental and human health.

    references

  • Wu, Y., Meng, Y., Zhang, H., Hao, L., Zeng, T., Shi, Y., Chen, Y., Qiao, N. and Ren, Y. (2025). Ecological and Health Risks of Polycyclic Aromatic Hydrocarbons in Particulate Matter in Chinese Cities. GeoHealth, [online] 9(6). doi:https://doi.org/10.1029/2024gh001126.

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

  • Activation: Uses outdoor rainwater and humidity to hydrate the freeze-dried system, so no manual activation is needed.
  • One-time use: Bacillus subtilis can use PAHs as a carbon source, so the system is designed to keep breaking down fresh pollutant deposits over repeated exposure.
  • Stability: The encapsulation layer in the paint matrix helps protect the enzymes from environmental exposures.

    • Homework question from Ally Huang

    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)

    Galactic cosmic radiation poses a major health risk to astronauts, causing DNA damage, cancer risk, neurocognitive decline, and cardiovascular disease (Chancellor et al., 2014). This challenge is critical as long-duration missions to Mars exceed current radiation safety limits. Addressing it is vital for enabling deep-space exploration and protecting crew health. Scientifically, it is compelling to investigate whether BioBits cell-free systems can both detect DNA-damaged biomarkers and respond by producing DNA repair enzymes on demand, offering a real-time response.

    references

  • Chancellor, J., Scott, G. and Sutton, J. (2014). Space Radiation: The Number One Risk to Astronaut Health beyond Low Earth Orbit. Life, [online] 4(3), pp.491–510. doi:https://doi.org/10.3390/life4030491.

    • 2. 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)

    γ-H2AX (phosphorylated histone H2AX) as a biomarker for cosmic radiation (Sethu et al., 2017) and the PARP-1 gene for DNA repair enzyme production (Ko and Ren, 2012).

    references

  • Ko, H.L. and Ren, E.C. (2012). Functional Aspects of PARP1 in DNA Repair and Transcription. Biomolecules, 2(4), pp.524–548. doi:https://doi.org/10.3390/biom2040524.
  • Hande, Mp., Zeegers, D., Venkatesan, S., Koh, S., Low, G.M., Srivastava, P., Sundaram, N., Sethu, S., Banerjee, B., Jayapal, M., Belyakov, O., Baskar, R. and Balajee, A. (2017). Biomarkers of ionizing radiation exposure: A multiparametric approach. Genome Integrity, 8(1), p.6. doi:https://doi.org/10.4103/2041-9414.198911.

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

    Galactic cosmic rays can cause severe DNA damage in astronauts’ immune cells, creating clustered double-strand breaks. Within about 30 minutes, this damage leads to the formation of γ-H2AX foci, which act as an early signal that DNA repair may be needed. By detecting γ-H2AX, a BioBits® cell-free system can be triggered to produce the repair enzyme PARP-1. PARP-1 helps coordinate the repair process, allowing the cell to fix DNA breaks before mutations start to build up. This kind of self-activating biosensor-and-response system is especially useful in deep space, where long resupply delays make it difficult to rely on traditional medicines. Instead, it provides on-demand DNA repair exactly when astronauts need it.

    references

  • Redon, C.E., Dickey, J.S., Bonner, W.M. and Sedelnikova, O.A. (2009). γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Advances in Space Research, 43(8), pp.1171–1178. doi:https://doi.org/10.1016/j.asr.2008.10.011.
  • Wang, Y., Luo, W. and Wang, Y. (2019). PARP-1 and its associated nucleases in DNA damage response. YDNA Repair, 81, p.102651. doi:https://doi.org/10.1016/j.dnarep.2019.102651.

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

    Goal:

    Create a toehold switch that senses γ-H2AX and turns on PARP-1 production in a BioBits® reaction. Then test whether the PARP-1 made this way can actually repair damaged DNA, as a proof-of-concept for a wearable radiation-response patch for astronauts.

    Reasoning:

    γ-H2AX appears quickly at sites where radiation causes DNA double-strand breaks, making it a reliable early warning signal. PARP-1 is one of the first proteins to respond to this damage, helping recruit the cell’s repair machinery. BioBits® cell-free systems have already been shown to work on the ISS, so combining them with toehold switches could create a self-contained system that detects DNA damage and immediately produces a repair protein without needing outside intervention. If small fragments of γ-H2AX can activate the toehold switch, this would demonstrate a detect and respond system for space radiation damage.

    references

  • Podhorecka, M., Skladanowski, A. and Bozko, P. (2010). H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy. Journal of Nucleic Acids, 2010, pp.1–9. doi:https://doi.org/10.4061/2010/920161.
  • Kinner, A., Wu, W., Staudt, C. and Iliakis, G. (2008). -H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research, 36(17), pp.5678–5694. doi:https://doi.org/10.1093/nar/gkn550.

    • 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)

  • Samples: Human lymphocytes exposed to simulated cosmic radiation and non-exposed controls.
  • >Methods: Use miniPCR to amplify PARP-1 DNA. Measure protein production and γ-H2AX signals with a P51 Fluorescence Viewer.
  • Data: Track fluorescence (protein expression), percentage of repaired DNA, and number of γ-H2AX foci before and after treatment.
  • Controls: Non-irradiated cells and a scrambled toehold switch.