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.
Cell free systems are much simpler to control because the reaction does not have to happen within a cell-wall, so scientists have direct control over concentrations, monitoring, and timing. Cell based production is much harder to control, since you need to control the expression and inputs of the cell. For example, if you need to do experiments to optimize production, or you need to produce proteins that would kill living cells.
Another benefit is that cell free systems can be freeze dried and transported. Living cells require water, which consist of 99% of volume in a biological reaction. Freeze drying allows for much lower cost of transport and storage, allowing for on-demand therapeutics, or rapid manufacturing.
Describe the main components of a cell-free expression system and explain the role of each component.
It is all the parts of the central dogma for protein expression:
- DNA: the template DNA for the protein you want to make
- DNA polymerase: Copies DNA
- RNA polymerase: DNA to RNA
- Ribosome: RNA to protein
These are contained in the cell extract.
We also need to add the other things that the cell would have generated for itself, like energy regeneration system.
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.
ATP is used by the reactions as an energy source, and energy is needed for any of the reactions to take place.
In the recitation, the method presented in purified system diagram uses creatine phosphate to convert ADP to ATP.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Prokaryotes are much simpler, with plasmid DNA, whereas eukaryotes can produce more complex proteins with post translational modifications. A simple protein like GFP could be a good candidate for prokaryotic systems, whereas eukaryotic systems could be a good candidate for IgG antibodies, which require post translational modification and are native to eukaryotes.
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.
The challenge for membrane proteins is that they are partially hydrophobic and could misfold in a water solution.
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.
A few possible reasons could be not enough energy, bottlenecking by transcription, or bottlenecking by translation.
We could measure the system, either by finding the amount of DNA, RNA, or ATP and plotting that over time in order to diagnose where the bottleneck comes from.
Homework question from Kate Adamala
Pick a function and describe it.
What would your synthetic cell do? What is the input and what is the output?
My synthetic cell would produce GFP based on the concentration of lactose in the system.
Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
Possibly, but a membrane makes it more ideal if it needs to be used in a biological system, otherwise the chemical concentrations would be diluted.
Could this function be realized by genetically modified natural cell?
Yes, it can be realized in a GMO cell.
Describe the desired outcome of your synthetic cell operation.
It will emit GFP and glow more intensely based on the concentration of lactose.
Design all components that would need to be part of your synthetic cell.
The membrane would be usual materials of phospholipids and cholesterol to make the membrane more stable, with a selective membrane pore to selectively allow lactose through to enable to biosensor.. Bacterial transaction and translation system is okay because the protein and function are simple.
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.)
LacY membrane protein to act as a pore for lactose, GFP expression protein, genes for Lac operon: LacI, LacZ to convert lactose to LacI-binding allolactose, LacO before the GFP gene to be repressed by LacI unless allolactose binds to LacI.
How will you measure the function of your system?
I can measure the amount of light produced in the presence of lactose to see if it is working.
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. How will the idea work, in more detail? Write 3-4 sentences or more. What societal challenge or market need will this address? How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?
Homework question from Ally Huang
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)
I’m curious about the impact of space on the body’s natural ways of healing. This will go towards understanding how space will affect our bodies and medicine.
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)
I’m interested in using spatial transcriptomics, which images the expressed proteins in space.
Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)
Using the same approach of the FISSEQ paper, I’m interested in seeing what genes are expressed during wound healing, but in the unique environment of space.
Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)
My hypothesis is that gene expression will be modified without gravity and wounds will heal slower as a result of those changes.
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 simulate wound healing using fibroblasts in space and on Earth, then use FISSEQ and compare the resulting expressed sequences using statistical techniques.