Week 9 — 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 not only is a robust and efficient system compared to in vivo methods, in which yields are comparatively lower, but if we go in terms of flexibility and experimental variables, there’s a lot of control to be had here given that we only use the components that are essential, this means that variables such as metabolism, possible toxicity, and laborous work such as transformation, transfection, just the whole genetically enginering part (and the assays that come with it, such as, verifying if my organism is indeed genetically modified!) is something to not worry about anymore. Now, because of all the previously mentioned, we do have more control too on elements needed for protein synthesis, such as, nucleotides, salts, DNA concentration, pH; said elements would typically be directed towards other cellular processes, but not here because there is no cell.

Now, as for two cases where it is more beneficial, one of the most obvious ones is the production of proteins that are normally toxic to the host. Since there’s no host, there’s no stopping in the production (hopefully). A second case would be when we need a quick check of a construct for something like a fluorescent protein, like I mentioned earlier, the protocols for genetic engineering do not need to be followed and so it is a huge time save.

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

Kind of like PCR, there are components that are a must, such as:

• DNA template
• An energy system
• Salts and buffers
• Aminoacids
• A cell lysate that comes with transcription and translation components such as enzymes and transcription factors

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.

Without energy, the machinery stops, all things in biology (unless I missed an exception just like biology always likes to have) do require energy.

The method I would use is glucose and phosphate to supply ATP, like the one in the article by Anderson, et al., (2015).

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

Prokaryotic are cheaper first and foremost, so I would choose a GFP for example. Now, everything changes depending on what you need, so, let’s say I need antibodies or I need a glicosilated protein, which can’t be done by prokaryotic systems; then I’d choose an eukaryotic system here, for.. let’s choose denosumab, If I recall correctly it is an antibody that targets osteoblasts and slows down osteoporosis.

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.

Membrane proteins and their folding problems thanks to the lack of membrane environment.. that’s the thing I’d tackle, the environment, so I’d add liposomes!

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.

Poor transcription: I’d check codon optimization or even promoter or DNA concentration (of course, I’d check all of these 1 by 1 otherwise I’m gonna be real confused at which one is it really) Protein misfolding: I’d add chaperones ATP running out: Add more components of my ATP regeneration system, or possibly just switch it to a different one

Homework question from Kate Adamala

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

Pick a function and describe it.

A biosensor for, let’s say mercury.

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

It would detect the presence of mercury! The input would be mercury, the output would be a fluorescent signal such as GFP.

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

Yes, it could be. Encapsulation’s preferred though because of stability.

    Could this function be realized by genetically modified natural cell?

Not by most, definitely. Because there are bacteria and fungi that are resistant to mercury thanks to detoxification proteins (Golysheva et al., 2025). But for the purpose of a system like this, it’d be optimal to not have to employ these proteins in the first place.

    Describe the desired outcome of your synthetic cell operation.

Fluorescence when mercury is present.

Design all components that would need to be part of your synthetic cell.
    What would be the membrane made of?

Phospholipids like POPC + cholesterol

    What would you encapsulate inside? Enzymes, small molecules.

Ribosomes, a DNA construct with a promoter that responds to mercury for GFP, aminoacids, an ATP regeneration system cell lysate components I’ve previously mentioned.

    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)

It could be just E. coli because there’s no eukaryotic PTMs needed

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

The mercury ions could diffuse in through the membrane

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.)

Lipids: POPC and cholesterol

Genes: GFP, merR regulator (https://www.uniprot.org/uniprotkb/P0A2Q8/entry) and mer promoter (https://registry.igem.org/parts/BBa_K346002)

    How will you measure the function of your system?

The intensity of GFP (it would be also neat to measure the intensity to different levels of mercury)

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.

A smart textile biosensor patch with embedded freeze-dried cell-free reactions that detects cancer proteins that are secreted through sweat.

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

The textile would contain freeze-dried cell free reactions integrated into this wearable patch and when it comes in contact with cancer proteins that come from sweat, the sweat would activate a reaction in the cell-free system, which would be the expression of a reporter, such as the generation of a visible color like dye or fluorescence.

What societal challenge or market need will this address?

Personal health monitoring, specifically, early cancer detection.

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

Sweat contains water (I think) or, well, it’s a liquid so that should be enough to activate the patch. It might be a one-time use so, I suppose multiple once every few months would be a good routinary way of going about it.

This idea was inspired by my molecular biology teacher, Dr. Azael Adrián Cavazos Jaramillo, who has made a biosensor with electrochemistry for cancer. Very interesting stuff!

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)

Radiation in space: it can damage DNA in long missions. In a distant future, this’ll be a problem because future exploration will depend on the safety, more like stability of biological materials that have DNA in it, without addressing this, DNA damage could happen and gene expression would change. A cell-free system here is useful because BioBits can express proteins without the need of living ccells, and the P51 viewer can detect fluorescent outputs quite simply.

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)

Multiple GFP-coding DNA sequences exposed to potential DNA-damaging conditions. Let’s let it sit on the moon for some time, or mars (simulating it’s under human skin)

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

If the GFP gene is damaged, the BioBits system may produce less fluorescent protein, or just simply, none at all, and so it makes it a simple reporter for seeing how DNA integrity (as in how much the DNA sequence remains unaltered) affects expression in a spacial context. Thanks to the function of the P51 viewer, then this would be a pretty straightforward readout, whether the damaged DNA still functions, or not. It’d also be interesting to sequence the genes once in Earth. Maybe in space if that’s possible too.

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

My hypothesis is that the DNA exposed to possible damaging conditions in space will produce a lower GFP expression or none at all in hte BioBits cell free system than intact DNA. I would expect intact GFP DNA to give a stronger, “default” fluorescent signal, while damaged DNA will give a weaks ingla because the sequence may not be able to be transcribed and translated as efficiently, or it simply just won’t be able to. The goal is to study the sensitivity of gene expression in DNA that’s in a spacial context. The relevance comes from one of humanity’s challenges: understanding space’s effect on DNA stability.

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)

I would prepare the BioBits reactions with two samples: intact GFP DNA sequences and damaged GFP DNA sequences. The intact DNA would be a positive control and the damaged one would be a negative control, miniPCR would be used to amplify before adding it ot the BioBits reaction. And after expression, the fluorescence of both controls would be measured with the P51 Molecular Fluorescence Viewer. The data collected would be fluorescence intensity, and also a sequencing of both controls.

Homework Part B: Individual Final Project

We’d like students to start exploring their final project in depth this week! Of your three Aims, for this week you should have at least Aim 1 decided and written down.

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
Submit this Final Project selection form if you have not already.
Begin planning how you will write your final project documentation based on these guidelines
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.
    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.)

Done in my final project page!

References

Anderson, M. J., Stark, J. C., Hodgman, C. E., & Jewett, M. C. (2015). Energizing eukaryotic cell-free protein synthesis with glucose metabolism. FEBS letters, 589(15), 1723–1727. https://doi.org/10.1016/j.febslet.2015.05.045

Golysheva, A. A., Litvinenko, L. V., & Ivshina, I. B. (2025). Diversity of Mercury-Tolerant Microorganisms. Microorganisms, 13(6), 1350. https://doi.org/10.3390/microorganisms13061350