Week 9 HW: Cell-Free Systems

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 systems have an advantage in “extreme” situations, while in-vivo occurs within cells that have to be kept alive. In taking the process outside of the cell, you can handle it more roughly i.e. freeze drying the system for long-distance transport, or making a system that can be kickstarted just by adding water (in remote locations). Cell-free systems also have more control because they’re synthetic, so you can determine how big the cell is and what exactly goes in it.

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

The components of a cell-free system are:

  • Cell Extract/Lysate – the internal components of the cell that contains molecular machinery/cofactors such as ribosomes, RNA polymerase, etc
  • tRNA – an RNA molecule needed to transcribe the genetic code
  • Membrane channels – for communication within/out of the cell
  • Plasmids/linear DNAs – containing the DNA sequence needed for the reaction
  • Nucleotides – the building blocks of RNA, aka ATP, GTP, CTP, UTP
  • Salts and buffer – needed to maintain pH (e.g. HEPES buffer)
  • Other enzymes/cofactors that aid in part of the process → Coenzyme A, 3-PGA, Spermidine, NAD etc
  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 is needed to run the reactions, and having a constant supply is crucial the longer the reaction goes. One way of continuously providing ATP is picking chemical processes that can replenish resources. This is part of why some cell-free reactions (like ribose NMP) are more efficient, because they can produce ATP which is used for both RNA synthesis and an energy source, allowing the reaction to continue for longer.

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

Prokaryotic cells differ from eukaryotic cells in components; notably, they lack membrane-bound organelles. For cell-free, they are the standard and are very low cost/high throughput. They’re a simple iteration of cell-free systems.

Eukaryotic cells are more complex and high cost, but can work with certain proteins and antibodies that are toxic to prokaryotic cells. All eukaryotic cells have an endoplasmic reticulum, which lets them represent functions like protein folding and post-translational modifications in cell-free 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.

I’d set it up similar to the lab process in Homework 11–with a standardized list of all components that go in (salt/buffer, enzymes, nucleotides, cofactors, etc) and vary input volume to observe how expression of the protein changes. A potential challenge could be in measuring the expression of the membrane protein (something I’m not as familiar with myself). This could be addressed by linking production of the protein to a more easily measurable protein (like GFP) to be produced as well.

  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.

Possible reasons and their solutions are

  • The pH of the system is off – vary the concentration of buffer to adjust the pH across different trials
  • Maturation time is not within the window I’m observing – observe protein expression across the entire range of time and look at relative difference in expression at different times
  • Protein expression does not match up to the range I’m looking at (ie a fluorescent protein with low brightness) – recalibrating equipment to a known control? Or attempt a trial with a similar protein to see if results change (ie with a different fluorescent protein)

Homework question from Kate Adamala – Design an example of a useful synthetic minimal cell as follows:

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

My synthetic cell can be designed for medical applications, such as in drug production. The input would be the template for the molecule, e.g. inserted as a plasmid, with the output being assembly of the target drug and potentially a light indicator for success.

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

The process needs to be isolated, so a membrane (and therefore synthetic cell) is necessary.

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

It could potentially, but the process of biomanufacturing generally needs to happen in a cleanroom or at least a clean space. A synthetic cell has more understood components and a more controlled environment within itself compared to a natural cell, and the process could be better standardized.

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

Ideally, given a plasmid containing the sequence of the drug, they are able to produce it and a fluorescent protein.

  1. Design all components that would need to be part of your synthetic cell.
    1. What would be the membrane made of? Phospholipids + cholesterol.
    2. What would you encapsulate inside? Enzymes, small molecules.

Cell-free Tx/Tl system, plasmid/DNA encoding drug, fluorescent protein, amino acids, nucleotides, other enzymes and coenzymes TBD

  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)

Bacterial (and probably E. coli) should be fine. I’m not currently aware of any further modifications that need to occur after translation.

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

I would likely need membrane channel pores to allow for molecules to cross over, especially for the output proteins.

  1. Experimental details
    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.)

Lipids: POPC (phospholipid), cholesterol Genes: fluorescent protein (e.g. GFP), E. coli, gene for channel pores

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

The results should be measured in fluorescence output.

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 propose that cell-free biosensors for pollutants found in acid rain could be integrated into rain jackets as a concept of wearable environmental sensors.

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

This idea builds upon detection of acid rain through pH testing, by incorporating the response of test strips into textiles. Rain jackets are already designed for a rainy environment. If additional cell-free layers were engineered on top of the waterproof layer, similar to what was discussed in class, we could design a rain jacket that could change color in response to low pH in rainwater. This could be done by joining the cell-free system with a fluorescent protein or other colorimetric protein that operates well in low pH. The end result would be a more “live” response to the individual’s local environmental contaminants, allowing the user to constantly evaluate their surroundings while also acting as a functional article of clothing.

  • What societal challenge or market need will this address?

This is intended to be a first step in responding to local environmental injustice, giving individuals more agency to observe and detect the extent of pollution in their backyard.

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

One concern is the possibility of overwhelming the cell-free systems with water, since rain jackets are generally worn longterm (whenever it rains). Additionally, the jacket might be a one-time use, since the detection of acid rain once would render it ineffective for the second rainfall. This could be addressed by intentional design, allowing only a limited amount of water to reach the cell-free system (i.e. smaller access holes for water droplets) and having a swappable cell-free system layer on the jacket.

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)

Quorum sensing is a method for bacteria to communicate on a large, colony-wide scale. This occurs through autoinducers, chemical molecules that diffuse or are transported between the cells. I think this is an interesting topic to apply to space, as testing these cells can reveal how the process of diffusion/osmosis is impacted by microgravity as well as opening a path up to explore different mediums of communication for biology in space.

  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)

Aliivibrio fischeri would be interesting as it’s a bacteria that provides bioluminescence within a squid once its population grows enough. The proteins involved are luciferase and LuxY.

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

Lots of prior proposals tackle the effects of microgravity, so this is sort of similar. The luciferase protein will get produced as a result of coordination across a bacteria colony. In getting to that result of fluorescence, we want to see how successfully the bacteria cells communicate with each other in microgravity.

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

I hypothesize that the proteins will have a faster response time and increased quorum sensing in spaceflight, as this seems to be the pattern with prior quorum sensing experiments. This might show itself in the form of fluorescence appearing much faster than it would on Earth, but might also be a false positive where fluorescence appears before cells have reached an appropriate density. This is actually a bit different than what I would have thought by intuition–I imagined that microgravity would make it harder for chemical molecules to travel across cells, which seems true, but in this case the settled molecules would be wrongfully interpreted as “positive” signals sent by other cells, resulting in a denser perceived network of cells.

  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)

The experiment is outlined below, though I’m a little skeptical of how easy it would be to implement:

  • To represent cell density, I would have a cell-free system be composed of the internals of Aliivibrio fischeri, including a way to generate and maintain autoinducers. The reaction could be carried out in several samples with different cell densities each.
  • The cell-free system would also include a plasmid that codes for luciferase, for when the switch to produce fluorescent protein is “flipped”.
  • Controls would require samples that are above and below the density needed for the bacteria to glow, based on experimental data gathered on Earth
  • Data would be detected value of fluorescence, which would need specific measurement tools.