Week 9 HW: CELL-FREE SYSTEMS
Homework Part A: General and Lecturer-Specific Questions
General Homework Questions
1. Advantages of cell-free protein synthesis
Cell-free protein synthesis is more flexible than traditional in vivo methods because we can directly control conditions like temperature, pH, DNA concentration, and energy supply without worrying about keeping cells alive. It is also faster since proteins can be produced in a few hours.
Two situations where cell-free systems are more useful are:
- Producing toxic proteins that would kill living cells
- Making membrane proteins or proteins that are difficult to express inside cells
2. Main components of a cell-free expression system
A cell-free system usually contains:
- Cell extract: provides ribosomes, enzymes, and machinery for transcription and translation
- DNA template: contains the gene for the protein we want to produce
- Amino acids: building blocks for proteins
- Energy source (ATP/GTP): powers transcription and translation
- Salts and cofactors: help enzymes work correctly
3. Why energy regeneration is important
Protein synthesis uses a lot of ATP, so energy can run out quickly in cell-free systems. Without ATP regeneration, protein production stops fast. One way to maintain ATP supply is by adding phosphoenolpyruvate (PEP) or creatine phosphate as an energy regeneration system. These molecules help continuously recycle ATP during the reaction.
4. Prokaryotic vs eukaryotic cell-free systems
Prokaryotic systems, like E. coli extracts, are faster and cheaper. They work well for simple proteins. For example, producing GFP in an E. coli system works well because GFP does not need many modifications.
Eukaryotic systems are better for complex proteins that need folding or post-translational modifications. For example, producing antibodies in a mammalian system requires proper glycosylation and folding.
5. Designing a cell-free experiment for membrane proteins
To express a membrane protein, I would add liposomes or detergents into the cell-free reaction so the protein has a membrane-like environment to fold correctly. The main challenge is that membrane proteins easily aggregate or misfold. To solve this, I would optimize temperature, use lower expression rates, and include lipid nanodiscs or liposomes.
6. Reasons for low protein yield
- Poor DNA quality or low DNA concentration: Check DNA purity and increase template concentration
- ATP depletion: Improve the energy regeneration system
- Protein degradation by proteases: Add protease inhibitors or reduce incubation time
Homework Question from Kate Adamala
1. Function of the synthetic cell
My synthetic cell would detect mercury contamination in water and produce a fluorescent signal.
- Input: mercury ions
- Output: green fluorescence
2. Could this work without encapsulation?
Not completely. Encapsulation is important because the reactions need a controlled environment and the fluorescent response should stay localized inside the synthetic cell.
3. Could this be done with a genetically modified natural cell?
Yes, bacteria could also be engineered to detect mercury. However, synthetic cells are safer because they cannot reproduce or escape into the environment.
4. Desired outcome
When mercury is present in contaminated water, the synthetic cells should glow green so contamination can be detected easily.
5. Components of the synthetic cell
The synthetic cell would contain: cell-free Tx/Tl system, GFP gene under a mercury-sensitive promoter, ribosomes, ATP, amino acids, and salts.
6. Membrane composition
The membrane would be made of phospholipids and cholesterol to keep the system stable.
7. What would be encapsulated?
Inside I would encapsulate: E. coli cell-free extract, GFP DNA, amino acids, ATP, and enzymes for transcription and translation.
8. Which Tx/Tl system?
A bacterial system from E. coli would be enough because GFP expression does not require mammalian modifications.
9. Communication with the environment
Mercury ions are small enough to diffuse through membrane pores, so the synthetic cell could sense them directly.
Experimental Details
Lipids and genes
- Lipids: POPC and Cholesterol
- Genes: GFP and MerR mercury-responsive regulator
Measuring the function I would measure fluorescence intensity using a fluorescence reader or microscope.
Homework Question from Peter Nguyen
Application Field: Textiles/Fashion
One-sentence pitch
I propose a smart fabric with freeze-dried cell-free sensors that detect sweat dehydration markers and change color when the user needs water.
How would it work?
The fabric would contain freeze-dried cell-free reactions embedded into small patches. When sweat activates the system, it detects salt concentration related to dehydration. The reaction would then produce a visible color or fluorescent signal. Athletes or workers in hot environments could quickly know when they are dehydrated.
Problem it solves
Dehydration is common in sports and outdoor jobs, and many people do not notice symptoms early enough. This textile could help prevent heat exhaustion and improve health monitoring.
Addressing limitations
To deal with stability issues, the reactions could stay freeze-dried until sweat activates them. The sensor patches could also be replaceable after one use.
Homework Question from Ally Huang
1. Motivation
Long space missions expose astronauts to radiation and microgravity, which can damage DNA and affect human health. Detecting DNA damage quickly in space is important because astronauts have limited medical resources. Using cell-free systems could provide a fast and portable way to monitor genetic damage during missions. This project is relevant for future missions to the Moon or Mars, where astronauts will spend long periods far from Earth.
2. Molecular target
DNA damage response genes, especially p53-related DNA repair pathways.
3. Relation to the problem
Radiation in space can damage DNA and increase mutation rates. Monitoring genes involved in DNA repair could help detect early biological stress in astronauts. Changes in DNA damage markers would show whether radiation exposure is affecting cells during space missions.
4. Hypothesis / research goal
My hypothesis is that radiation exposure in space increases activation of DNA damage response pathways, which can be detected using cell-free systems. I want to test whether BioBits® reactions can identify DNA damage biomarkers quickly and reliably in microgravity conditions. If successful, this system could become a portable diagnostic tool for astronaut health monitoring during long missions.
5. Experimental plan
I would compare DNA samples exposed to radiation with non-exposed control samples. The samples would be amplified using miniPCR®, then analyzed using BioBits® cell-free reactions linked to fluorescent reporters. Fluorescence intensity would be measured using the P51 Molecular Fluorescence Viewer. Increased fluorescence would indicate activation of DNA damage-related targets.