Week 9 HW: Cell Free Systems

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.

In cell-free protein synthesis, variables such as pH, ion concentration, components, etc. are more adjustable. Additionally, researchers can add or remove components at any time. Two cases where cell-free expression is more beneficial than cell production are when the protein is toxic or when large amounts of protein are needed quickly.

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

ATP (energy source), DNA (template for transcription and translation), nucleotides (for mRNA synthesis), ribosomes and tRNAs with amino acids (for translation), RNA polymerase (for translation).

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 provision regeneration is critical because ATP is required both for RNA synthesis and for protein synthesis (formation of phosphodiester and peptide bonds). One method that could be used is to add phosphoenol pyruvate (the precursor to pyruvate in glycolysis) and pyruvate kinase to produce ATP as the cell-free system runs.

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

Prokaryotic cell-free expression systems provide very high yield and quick reactions, but eukaryotic cell-free expression system allow for proper folding of more complex proteins. Eukaryotic cell-free systems may also be able to add post-translational modifications to proteins or insert proteins into membranes (microsomes, which are essentially vesicles).

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/weak promotor or RBS. Troubleshoot by changing the promoter/RBS to something that is known to be stronger.
• Running out of energy/resources. Troubleshoot by adding more ATP, PEP, and pyruvate kinase or more nucleotides and amino acids.
• Protein misfolding. Troubleshoot by lowering the temperature to slow the reaction or add chaperone proteins.

Homework Question from Kate Adamala

1. Pick a function and describe it.

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

My synthetic cell would assemble a phage. The input would be a phage’s genome, and the output would be phage proteins that would (hopefully) assemble into a working phage. Perhaps I would also need to add some bacterial chaperone proteins that I determine are necessary for phage assembly.

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

Yes, this could be realized by cell-free Tx/Tl alone because no phage proteins are membrane-bound.

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

Yes, that is essentially phage rebooting, where the phage’s genome is electroporated into its host, and it replicates as usual.

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

A working phage, so essentially a lysate that can be used to infect the host and create more phages.

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

a. What would be the membrane made of?

It doesn’t really matter since phage proteins don’t depend on a membrane to fold.

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

Nucleotides, amino acids, ATP, RNA polymerase, ribosomes, phage genome.

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 is enough because phages infect bacteria and use their machinery.

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

I’m not sure that my synthetic cell would have to communicate with the environment. Perhaps it would still need channels to import macromolecules/nutrients, but everything to make a phage would be contained within the cell.

4. 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.)

Genes: capsid proteins, tail proteins (tape measure, tail tube, tail fibers), terminase, head-to-tail adaptors/stoppers, etc.
Lipids: as far as I know, nothing specific is necessary, so phospholipids are enough.

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

I would conduct plaque assays to calculate the titer of the lysate made by the synthetic cells.

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.

Cloth that doesn’t let oil stains set by releasing an emulsifier when run under water.

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

One problem I envision encountering is that an emulsifier (which detergents are) kill cells, so how could a cell produce an emulsifier? Perhaps it would be possible for a cell to secrete the parts of an emulsifier—the hydrophobic part and the hydrophilic part—separately. Then, due to the extracellular conditions outside the cell being different from those inside the cell, the hydrophobic and hydrophilic part could self-assemble outside of the cell, so it doesn’t kill the cell as it’s being made. This emulsifier would not spread very far in the fabric, so cells from the surrounding area would eventually grow back over the cells that were killed when the emulsifer was released.

What societal challenge or market need will this address?

Clothing sustainability. It is very difficult to get oil/grease stains out of clothing if the stain is not washed immediately, so stained clothing gets thrown out. If it is possible to make it so that the stain is easier to wash out, more clothes would last longer.

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

I’ve already addressed this a bit in the second question, but another limitation is making sure the cells have enough resources to continue living and making the emulsifier.

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)

During space travel, there are limited resources, as additional weight can inhibit take off of a rocket. Additionally, while in outerspace, astronauts’ immune systems are dampened. As such, they are more susceptible to infections. This means that it would be helpful to have a way to produce medicines, such as common antibiotics, on demand.

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)

Amoxicillin. This is a commonly used antibioitic used to treat ear, sinus, throat, and pneumonia infections.

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

Ear, sinus, and throat infections along with pneumonia are common infections that may happen in space. It is possible that there are no medications to eradicate the bacterial pathogen causing these infections, so having a way to produce it on demand in space would be helpful.

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

This project aims to produce amoxicillin in vitro using a cell-free expression system. I hypothesize that it may be possible to produce the enzymes involed in the synthesis pathway using cell-free expression, then use those enzymes under the right conditions with the right starting materials to synthesize amoxicillin in space.

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 use separate cell-free expression systems to synthesize each of the enzymes in the amoxicillin synthesis pathway. I can determine that the enzymes have been synthesized correctly by running something like a western blot using an antibody if it exists. Then, I would combine the reagents necessary for amoxicillin synthesis with the sythesized enzymes. It is possible that some enzymes cannot be in the same tube together, in which case a separation technique would be necessary to isolate the amoxicillin intermediate and transfer it to the tube with the next enzyme. I would work to limit the number of times I’d have to do this, as that would decrease the yield of amoxicillin each time. I would assess whether amoxicillin had been correctly synthesized by the end of the procedure using a chemistry technique such as high-performance liquid chromatography (de Marco et al., 2017).