Week 9 HW: Cell Free Systems
๐ Week 9 Homework ๐
Homework Part A: General and Lecturer-Specific Questions
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 protein synthesis is basically taking the protein-making machinery out of a living cell and running the process in a test tube. The biggest advantage over traditional in vivo methods (which use living cells) is that you do not have to worry about keeping a cell alive. When you work with living cells, the cell membrane blocks you from easily changing the environment, and the cell’s natural life-cycles get in the way.
By removing the cell entirely, you get an open system. This gives you much more flexibility and control over the experimental variables because you can easily add, remove, or change ingredients in the test tube without worrying about burdening the cell.
Because of this level of control, cell-free expression is much more beneficial than using live cells in a few specific cases like producing toxic proteins, If a protein is naturally toxic, trying to grow it in a living cell will just kill the cell before the process is finished, unlike cell-free systems that don’t have a life to lose, so it can successfully produce it, another example is using unnatural ingredients, If you want to build a protein using artificial or unnatural amino acids, living cells will usually reject them. In a cell-free test tube, you can easily force the system to accept and use these custom parts.
Describe the main components of a cell-free expression system and explain the role of each component.
A cell-free expression system is made up of five main components that work together to produce a protein outside of a living cell:
Cell Extract: This is the biological “machinery” (like ribosomes and enzymes) harvested from crushed cells. Its role is to actually read the instructions and assemble the protein.
Genetic Template (DNA or RNA): This acts as the blueprint. It provides the specific instructions for which protein the machinery needs to build.
Amino Acids: These are the raw materials or building blocks that the machinery links together to form the final protein.
Energy Source: The process requires power to run.
Reaction Buffer: This is a mixture of salts and chemicals (like magnesium) that maintains the correct pH and environment.
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 regeneration is critical in cell-free systems because making proteins takes a massive amount of energy. If I just add a starting dose of ATP (the fuel) to my test tube, my experiment will fail quickly for two reasons:
I will run out of fuel almost instantly, causing the reaction to stop.
The “dead” fuel (ADP) builds up in the test tube and actually acts like toxic exhaust, slowing down the machinery.
To make sure I have a continuous ATP supply in my experiment, I would use an energy regeneration system like creatine phosphate and creatine kinase. I like to think of this as a backup battery charger. I would add the high-energy chemical (creatine phosphate) and a specific enzyme (creatine kinase) to my test tube. As my machinery burns through the ATP and turns it into dead ADP, the enzyme automatically uses the creatine phosphate to recharge the ADP back into fresh ATP, keeping my experiment running smoothly.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
When comparing cell-free expression systems, the main difference comes down to speed, cost, and the complexity of the protein we want to make.
Prokaryotic Systems (e coli): These are fast, cheap, and produce high yields of proteins. However, they lack the machinery to properly fold complex proteins or add necessary chemical modifications.
Eukaryotic Systems (yeast): These are slower and more expensive, yielding less protein. But, they contain the advanced machinery needed to perfectly fold complex proteins and add necessary modifications (like attaching sugar molecules).
My Protein Choices:
For the Prokaryotic System: i’d choose to produce Green Fluorescent Protein (GFP), because GFP is a very simple, standard protein that folds easily on its own. Since it does not require complex modifications, we can take advantage of the prokaryotic system’s speed and cheap cost to produce massive amounts of it quickly.
For the Eukaryotic System: I’d choose to produce a Human Antibody, since Antibodies are highly complex proteins that require intricate folding and specific chemical modifications (like glycosylation) to actually work. A simple bacterial system would build it incorrectly, so we must use the more advanced eukaryotic system to ensure it functions properly.
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.
When designing a cell-free experiment to produce a membrane protein, the biggest challenge we face is that these proteins are highly hydrophobic (water-repelling). Because a standard cell-free reaction mixture is mostly water-based, newly made membrane proteins will naturally clump together and fold incorrectly to hide from the water, becoming completely useless.
To optimize our setup and solve this problem, we need to provide a fatty environment directly in the test tube so the protein has somewhere to go as soon as it is built. We would design the experiment by adding either Liposomes which are artificial bubbles of cell membrane into the liquid. As the machinery builds our membrane protein, it will insert it directly into the liposome wall, mimicking a natural cell, another option is detergennts, We can use specific detergents that wrap around the protein to shield it from the water, keeping it dissolved and properly folded.
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.
If we observe a low yield of our target protein in our cell-free system, it can be either
Reason 1: Our genetic template is degraded. The DNA or RNA instructions might be breaking down before the machinery can read them, and to solve it we would use higher-quality, purified DNA/RNA and add special chemicals (like RNase inhibitors) to the mix to protect the blueprint from being destroyed.
Reason 2: We ran out of energy or building blocks. The system might have burned through all its ATP or used up all the amino acids, here we would add an energy regeneration system (like creatine phosphate) to keep the power on, or by adding a fresh batch of amino acids mid-reaction.
Reason 3: The buffer conditions are wrong. The environment in the test tube, specifically the salt or magnesium levels, might not be ideal for our specific protein, We would set up a few small, test reactions with slightly different magnesium concentrations to find the exact “sweet spot” where our protein grows best.
Homework question from Kate Adamala
Function and Description
I am designing a synthetic cell that acts as an environmental sensor for contaminated drinking water.
Input: Arsenic.
Output: Red Fluorescent Protein (RFP).
Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
No, if we just poured our raw cell-free protein machinery directly into a river or a cup of tested water, the machinery would instantly dilute into the water and be destroyed by the environment. The cell membrane acts as a protective shield, keeping our machinery concentrated and safe in its own little bubble (very similar to the example answer given in homeowrk)
Could this function be realized by a genetically modified natural cell?
Yes, but there is a major flaw with living cells here: arsenic is highly toxic. If the water sample is heavily contaminated, the arsenic will likely kill the living bacteria before they even have a chance to produce the red warning color. Because our synthetic cell is basically “dead” chemical machinery, it cannot be killed by the toxin.
Desired Outcome of Operation
When we drop our synthetic cells into a water sample, they will glow bright red if the water is contaminated with arsenic, acting as a clear visual warning.
Design Components
The Membrane: We will use a basic mix of POPC (a standard, easy-to-use lipid) and cholesterol to make the bubble stable.
Inside the Cell (Encapsulation): We will pack it with our cell-free Tx/Tl machinery, amino acids, and the specific genetic instructions (DNA) needed to sense arsenic and build the red protein.
Tx/Tl System Origin We will use a Bacterial (E. coli) system since bacterial systems are much cheaper and easier for a basic sensor like this.
Communication with the Environment Because arsenic ions are very small, we will rely on them being naturally permeable to our simple lipid membrane. The arsenic will slip through the fat bubble into the inside. The output (the red protein) is too big to escape, so it stays trapped inside, making the entire bubble light up red.
Experimental Details
Lipids: POPC and Cholesterol.
Enzymes: E. coli cell-free extract (the Tx/Tl machinery).
Genes: We will use the Pars promoter and arsR gene (genetic switch that reacts to arsenic) connected to the mCherry gene (RFP).
Measurement We will measure the function of our system using a fluorometer (a machine that measures specific colors of light) to track how much red fluorescence is being produced. For a quick visual check, we could even just shine a UV flashlight on the sample to see if it glows red!
Homework question from Peter Nguyen
Idk why but my idea kinda reminds me of Detroit Become Human XD
Infection-Detecting Smart Bandage
We are developing a smart, wearable bandage that uses freeze-dried cell-free sensors to instantly change color when it detects an early infection in a wound.
How the Idea Will Work
We will embed freeze-dried, dormant cell-free machinery directly into the cotton fibers of a standard bandage. When a person puts the bandage on a cut, the natural moisture from the wound (blood or plasma) will soak into the fabric and “wake up” the machinery. If the cut becomes infected, the harmful bacteria will release specific chemical toxins. Our built-in cell-free sensors are programmed to detect those specific toxins; if they find them, the machinery will produce a brightly colored protein, turning the bandage blue to warn the user of the infection.
The Societal Challenge and Market Need
This directly addresses the massive global problem of delayed medical care and severe infections. Right now, most people do not know a wound is infected until it becomes hot, swollen, and dangerous, often requiring heavy antibiotics or a hospital visit. Our smart bandage gives patients an easy, visual warning at home or disaster araes so they can get a simple treatment before the problem gets worse.
Addressing the Limitations of Cell-Free Systems Cell-free systems have limits, but we can actually use them to our advantage in this design:
Activation with water: The reaction stays completely paused on the shelf and is only activated by the natural moisture of the wound itself (unfortunately water can provide perfect medium for bacteria to grow).
Stability: To keep the freeze-dried machinery stable for months or years, we simply package our smart bandages in airtight, moisture-proof foil wrappers (just like normal sterile bandages).
One-time use: A cell-free reaction eventually runs out of energy and stops, meaning it only works once. However so does normal bandages :D
Homework question from Ally Huang
After having a discussion of the idea with Gemini, i was lazy :( and had it do the writing instead because i was really busy sooo i am sorry for that XD
On-Demand Parathyroid Hormone Production
Background Information (Maximum 100 words)
Astronauts lose a dangerous amount of bone mass during long spaceflights due to zero gravity. Taking pre-made liquid medicines from Earth is difficult because they are heavy, take up precious cargo space, and expire quickly due to harsh space radiation. We need a way to manufacture fresh medicines directly on the spaceship. This is scientifically interesting and absolutely critical for keeping astronauts healthy on long missions to Mars where supply drops are impossible.
Molecular or Genetic Target (Maximum 30 words)
The target is the gene for Parathyroid Hormone (PTH), which is a specific protein that tells the human body to build new bone mass.
Relation to Space Biology Challenge (Maximum 100 words)
PTH is a well-known protein medicine used on Earth to treat severe bone loss. By targeting the PTH gene, we can use the BioBits cell-free system as a mini medical factory. Instead of bringing fragile, expiring liquid medicine into space, we just bring the lightweight, freeze-dried DNA instructions for PTH. When astronauts need the medicine, they simply add water, and the BioBits system builds the fresh PTH protein on demand to treat their decaying bones.
Hypothesis and Research Goal (Maximum 150 words)
Our goal is to prove that a freeze-dried cell-free system (BioBits) can successfully manufacture a complex human medicine (PTH) in a zero-gravity environment. We hypothesize that the microgravity on the space station will not stop the cell-free machinery from correctly reading the DNA and assembling the protein. Because cell-free systems are just chemical reactions and do not rely on living cells (which often get stressed or behave unpredictably in space), we believe the protein-building process will work just as perfectly in space as it does on Earth.
Experimental Plan (Maximum 100 words)
On the space station, we will add water to two sets of freeze-dried BioBits tubes and warm them using the miniPCR machine.
Test Sample: BioBits containing the DNA instructions for the PTH bone medicine.
Positive Control: BioBits containing DNA for a red fluorescent protein.
After a few hours, we will look at the control tube using the P51 Viewer; if it glows red, we know the BioBits machinery survived the trip and is working. Finally, we will test our main sample using a simple protein test strip to confirm the fresh PTH medicine was successfully built.
Homework Part B: Individual Final Project
