Week 8 HW: Cell-Free Systems

General and Lecturer-Specific 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.

There are many advantages of cell-free protein synthesis over traditional in vivo methods. Cell-free systems allow you to have more control over the conditions of your experiment (ie. DNA, proteins, small molecules, etc.). Unlike working in vivo methods, where things are a bit more of a “black box”.

Two cases where cell-free is more beneficial:

i. Cell-free is more beneficial when it comes to rapid prototyping of metabolic pathways. In the lecture it was mentioned that a four-enzyme pathway would have taken weeks of cloning and transformation with an in-vivo method could take about an hour if done in a cell-free system.

ii. Cell-free systems can be more affordable. For example, there are cell-free freeze dried systems that cost under a dollar and can therefore be used in low-resource communities to detect Ebola, Zika or other diseases.

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

A cell-free expression system essentially mimics what is going on in a bacteria. You scoop out the ribosomes, polymerases, the tRNAs - and then you components like amino acids and an energy source.

Below are the main components in detail:

DNA template - the DNA for the protein you want to make. Amino acids - the building blocks for protein synthesis. tRNAs - small RNA molecule that decodes mRNA and delivers amino acids to ribosome. Energy source - something that powers the transcription and translation (could be ATP regeneration) Cell extract - This is what includes the ribosomes and RNA polymerases (PURE express is a version of this)

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.

Transcription and translation cost energy. In a cell-free system the reaction quicky uses up either the ATP or GTP - it’s designed more for a one time use. To avoid this there are ways you can create a regenerative ATP supply. One of the ways to create this is to use the Phosphoketolase (PKT) Systems: these use a pathway of enzymes to turn phosphorylate ADP into usable ATP.

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

A prokaryotic cell-free expression system is fairly straightforward, it can be E.coli-based which is already highly understood. It is high-yield and cheaper. You could produce GFP in this system and it would be quick and efficient.

A eukaryotic cell-free system is useful if you need post-translational modifications. There are extra components in eukaryotic cells like the endoplasmic reticulum that attaches sugar chains to the proteins being expressed. There are also chaperones that help the protein fold correctly. It more expensive and has a lower yield than prokaryotic cell free system.

5. 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.

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.

Homework question from Kate Amadala

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

1. Pick a function and describe it.

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

I would want to make a synthetic cell that would detect heavy metals in the soil (inspired by my earlier homeworks about Schewanella and Geobacter bacterias). The input would be the heavy metal molecules that would diffuse into the cells. The output would be a chromoprotein or maybe GFP that would be expressed if the heavy metals were detected.

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

It could be. Without encapsulation the cell-free system is more one-shot (not reusable). So it would functionally still work, but it would be more useful if the cell-free system had a membrane to better protect it from the soil elements. Although technically it’s supposed to be used as more of a one shot because once the GFP proteins are expressed you can’t reuse it. But you might want the sensor to last a while until it does detect something.

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

Totally. E.coli and Shewanella have been genetically modified with metal-detecting promoters to respond to heavy metals. Of course, when working with natural cells you have to make sure that the environmental conditions are perfect in order to keep the natural cell alive.

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

The idea would be to mix these freeze-dried synthetic cells with soil and water. And then if heavy metals are detected the cells will change color. In the case of using GFP you will have to flash blue or UV light in order to detect the change in color. There could be a variety of synthetic cells that change different colors based on different toxic metals. Since there’s a very limited biosecurity risks with the natural cells, these detectors could be used by non-scientists (like farmers) to detect issues with their soil.

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

a. What would be the membrane made of?

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

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 promotors, like Tet-ON, you need mammalian)

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

Homework question from Peter Nguyen

Summary Pitch: The development of freeze-dried cell-free systems that are embedded into building materials and are activated by water that seeps through the cracks in ceilings or wall, which in turn produces urease enzyme to start a biocementation process - to seal the crack before it spreads.

The freeze-dried cell-free systems will be mixed in with the concrete, so that there are cell-free systems a little everywhere in the mixture. When water touches the freeze-dried systems the production of the urease systems begins. This breaks down urea (also in the material) into ammonia and CO2. This raises the pH and provides carbonate which then bonds with calcium in the material to create limestone (calcium carbonate). Which seals the crack in the material.

This addresses a common contruction issue that affects many urban areas. Building materials are constantly degrading due to environmental issues so this will help the longevity of materials.

The cell-free solution for this issue does have a big limitation in that it is a one-time use. Using freeze-dried cell free systems to create self-healing concrete will not create a perpetually self-healing material. If there is a recurring water issue that causes a crack then the cell-free system will increase the longevity of the material but not solve the issue permanently.

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.

A challenge with space biology is the fact that DNA can degrade when exposed to the high levels of cosmic radiation that exist outside of Earth’s atmosphere. There is no easy way to detect how much radiation is affecting the DNA of astronauts. We could use the Biobits cell-free system to test DNA that has been exposed to radiation and compare it to regular control DNA to get a better understanding of how the astronauts are being impacted.

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)

The integrity of DNA templates will be studied by encoding for GFP after exposure to space radiation (and comparing with the control DNA), using fluorescence output from BioBits cell-free expression as the readout.

Homework Part B: Individual Final Project

Updated on the Individual Final Project page