Week 09 HW: Cell-Free Systems

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.

Advantages: cell-free systems allow for precise control over the reaction conditions, such as temperature, pH, and the concentration of substrates and cofactors. This can lead to higher yields and faster protein production compared to in vivo methods.

Examples:

• Production of toxic proteins that would harm living cells.
• Rapid prototyping of genetic circuits without the need for transformation and cell growth.

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

• Cell extract: contains the necessary machinery for transcription and translation, including ribosomes, tRNAs, and enzymes.
• Energy source: provides the necessary energy for protein synthesis, such as ATP or GTP.
• DNA template: contains the genetic information for the protein to be synthesized.
• Substrates: amino acids and nucleotides required for protein synthesis.
• Cofactors: molecules that assist in enzymatic reactions, such as magnesium ions.
• Buffer: maintains the optimal pH and ionic strength for the reaction.

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.

The synthesis of proteins requires ATP supply.

Phosphoenolpyruvate (PEP): it can be converted to pyruvate by the enzyme pyruvate kinase, which generates ATP from ADP in the process.

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

Prokaryotic: high yields, high speed, and low cost. Suitable for simple proteins without post-translational modifications (e.g. Green Fluorescent Protein GFP).

Eukaryotic: can perform post-translational modifications, but slower and more expensive. Suitable for complex proteins that require PTM (e.g. human insulin).

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.

Membrane proteins are hydrophobic (i.e. lipophilic). Therefore, I would use a cell-free system that mimcs the membrane lipid environment to avoid misfolding and aggregation. In specific, I would use a cell-free system that includes liposomes to create a suitable environment.

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.

• DNA tempalte degraded: replace the DNA template with a fresh one
• Insufficient energy supply: add more energy source (e.g. ATP or PEP)

Homework question from Kate Adamala

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

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

I would design a synthetic cell that can detect viral pathogens in water samples. The input would be the water sample, and the output would some kind of signal. For example, the cell could produce luciferase in response to the presence of viral RNA, which would emit light that can be easily detected.

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

Yes.

The cell-free system can be designed to include the necessary components for transcription and translation, as well as a reporter gene that produces a detectable signal in response to the target viral RNA.

Encapsulation is not needed for this function, as the cell-free system can operate in solution.

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

Yes. For example, based on Noctiluca scintillans.

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

Sensitive and low-cost detection of viral pathogens in water samples.

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

a. What would be the membrane made of?

Lipid bilayer.

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

Enzymes for transcription and translation, a reporter gene (e.g. luciferase), substrates (luciferin) and cofactors.

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)

Bacterial system is sufficient for this application.

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

The viral RNA can enter the cell-free system through pores in the membrane, and the luciferin substrate can also diffuse into the system to enable the luciferase reaction.

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: phosphatidylcholine, phosphatidylethanolamine, cholesterol.

Genes: RNA polymerase, luciferase gene.

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

Adding samples of viral RNA and measure the luminescence.

Homework question from Peter Nguyen

1. Write a one-sentence summary pitch sentence describing your concept.

A cell-free system integrated into textiles that can sense environmental pollutants and change color to alert the user.

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

The cell-free system will be designed to detect specific pollutants, such as heavy metals or viral pathogens. When the target pollutant is detected, the system will trigger a colorimetric reaction that changes the color of the textile. This could be achieved by incorporating a reporter gene that produces a pigment in response to the pollutant. The textile could be used in clothing or facial masks to provide real-time detection capabilities for human.

3. What societal challenge or market need will this address?

Not reusable: the color change is irreversible, so the textile would need to be replaced after each use. This could be addressed by designing a system that can be reset or by using a reversible color change mechanism.

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

To address the limitation of one-time use, I would design the system to be reversible. The color would change back when the pollutant is no longer detected.

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/ .

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)

In space, the intense radiation can cause genetic variation and protein misfolding, leading to reduced functionality and potential health risks for astronauts. A cell-free system could be used to produce functional proteins in space, mitigating these issues and supporting long-term space exploration.

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 molecular target I propose to study is the DNA repair enzyme, specifically the protein RAD51, which plays a crucial role in repairing DNA damage caused by radiation.

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

RAD51 is essential for repairing DNA double-strand breaks, which are a common form of damage caused by space radiation. By studying RAD51 in a cell-free system, we can understand how it functions under space-like conditions and potentially enhance its activity.

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

Hypothesis: Enhancing the activity of RAD51 in a cell-free system can improve DNA repair efficiency under space-like conditions.

5. 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 will use a cell-free system to express RAD51 and expose it to simulated space radiation. I will measure the DNA repair efficiency by assessing the ability of RAD51 to repair induced DNA damage using a reporter assay. Controls will include a cell-free system without RAD51 and a system with a known DNA repair-deficient mutant of RAD51.