This week introduces synthesis of proteins using cellular machinery outside of a cell.
Homework
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.
The biggest advantage of cell-free systems is that they offer an open environment where you have total control over experimental variables like pH and salt concentrations without a cell membrane getting in the way. This flexibility is especially beneficial when producing antimicrobial peptides or lysis proteins that would normally kill a living host, as well as for high-throughput screening of genetic circuits where you need to test many DNA variants in hours rather than waiting days for cultures to grow.
Describe the main components of a cell-free expression system and explain the role of each component.
A standard system essentially needs three main parts to function properly. The cell extract acts as the hardware, providing ribosomes and tRNAs, while the Energy Mix serves as the fuel by providing ATP and secondary sources like PEP. Finally, the DNA template works as the software instructions that contain the specific gene sequence you want to express in the tube.
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 absolutely critical in cell-free systems because once the initial ATP is used up, the synthesis stops since there is no active metabolism to recharge it like in a living cell. To ensure a continuous ATP supply during your experiment, you can use an enzymatic system such as creatine phosphate and creatine kinase to constantly convert ADP back into ATP while the reaction is running.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Prokaryotic systems are generally fast and high-yield but struggle with complex folding, whereas eukaryotic systems are slower but capable of post-translational modifications. For a prokaryotic setup, I would produce GFP because it gives a fast and simple fluorescence readout, but for a eukaryotic system, I would choose human insulin because it requires specific disulfide bonds that bacteria usually get wrong.
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.
Designing a cell-free experiment for membrane proteins is a challenge because these proteins are hydrophobic and tend to clump up in a liquid environment. To address this in my setup, I would add nanodiscs or liposomes to the reaction to provide a synthetic lipid bilayer where the protein can fold correctly as it is being synthesized, effectively mimicking its natural cellular environment.
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 I observe a low yield, it could usually due to three main issues that require a specific troubleshooting strategy. First, if the DNA template is degraded or salty I should re-purify it and check its integrity on a gel. Second, if the Magnesium (Mg^2+) levels are off for my specific extract I should run a titration assay to find the optimal concentration. Finally, to prevent RNase contamination from destroying my mRNA I should always add RNase inhibitors and use strictly nuclease-free reagents.
Homework question from Kate Adamala
Design an example of a useful synthetic minimal cell as follows:
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 act as a specific biosensor for mercury (HgĀ²āŗ) in water. The input is the mercury ions present in the environment, and the output is the release of a small signaling molecule called AI-2 (Autoinducer-2). This AI-2 then acts as a signal for a secondary population of natural bacteria to trigger a visible response.
Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
Not effectively. Without encapsulation, the AI-2 signal would be released immediately into the medium regardless of the mercury’s presence. The vesicle acts as a “logic gate” that keeps the signal locked inside until the mercury triggers the production of a membrane pore to release it.
Could this function be realized by genetically modified natural cell?
Yes, I could engineer a bacterium to do this, but natural cells often have cross-talk with other metals or metabolic burdens that affect the sensor. Using an SMC allows us to create a “clean” sensor that doesn’t die from the mercury toxicity and only responds to that specific input.
Describe the desired outcome of your synthetic cell operation.
When placed in a contaminated water sample, the SMCs detect mercury and release AI-2. This signal then turns a reporter colony of E. coli bright blue, providing a clear visual warning of the contamination
Design all components that would need to be part of your synthetic cell.
What would be the membrane made of?
I would use a mix of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) and cholesterol to ensure a stable, fluid membrane that mimics a natural cell but stays durable for field testing.
What would you encapsulate inside? Enzymes, small molecules.
I would encapsulate a bacterial cell-free Tx/Tl system (like PURE), a high concentration of the signaling molecule AI-2, and the DNA plasmid containing the mercury-responsive circuit.
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)
A bacterial system is perfect here because the mercury resistance operon (mer operon) is native to bacteria and works very efficiently with bacterial ribosomes and polymerases.
How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)
The mercury ions are small enough to diffuse through the lipid bilayer or through small constitutive pores. Once inside, they trigger the expression of a larger membrane channel that finally lets the encapsulated AI-2 escape into the surrounding environment.
Experimental details
List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying āsmall molecule membrane channelā pick the actual gene.)
Genes: I would use the merR regulatory gene and the merRo/p promoter to control the expression of alpha-hemolysin (hlyA) from Staphylococcus aureus, which will form the output pores.
Biological cells: Reporter E. coli strain LSB001 which is engineered to respond to AI-2 by producing chromogenic proteins.
How will you measure the function of your system?
I would measure the blue color intensity of the reporter bacteria using a standard plate reader or even just a smartphone camera for a qualitative field test. For more precision during development, I could co-encapsulate a fluorescent dye like calcein to track the exact rate of pore formation and signal release via microscopy.
Example solution
Based on: Lentini, R. et al., 2014. Nat comm, 5, p.4012.
Pick a function and describe it.
What would your synthetic cell do? What is the input and what is the output? Expand the sensing capacity of bacteria. Input: theophylline (inert to bacteria). Output of the SMC: IPTG. Output of the whole system: GFP produced in bacteria.
(Theophyline aptamer reference: *Martini, L. & Mansy, S.S., 2011. Cell-like systems with riboswitch controlled gene expression. Chemical Communications, 47(38), p.10734.*)
Could this function be realized by cell-free Tx/Tl alone, without encapsulation? No. If the IPTG were not encapsulated, it would go into the bacteria without the need of theophylline-induced membrane channel synthesis, thus the synthetic cell actuator would not exist.
Could this function be realized by genetically modified natural cell? Yes, in this particular case: the theophylline aptamer could be incorporated into a transformed gene. This lacks generality though ā it is easier to make SMC than modify bacteria, so in this system a single bacteria reporter can be used to detect various small molecules.
Describe the desired outcome of your synthetic cell operation. In the presence of SMC, bacteria sense theophylline.
Design all components that would need to be part of your synthetic cell.
What would be the membrane made of? Phospholipids + cholesterol.
What would you encapsulate inside? Enzymes, small molecules. cell-free Tx/Tl system, IPTG, gene for membrane transporter under the control of theophylline aptamer.
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, because of the theophylline riboswitch used as SMC input.
How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?) The membrane is permeable to the input molecule (theophylline), the output is IPTG that will cross the membrane via the membrane pore created after theophyline-initiated gene expression.
Experimental details
List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying āsmall molecule membrane channelā pick the actual gene.)
Lipids: POPC, cholesterol
Enzymes: bacterial cell-free Tx/Tl
Genes: a-hemolysin (aHL) to encapsulate in SMC
Biological cells: *E.coli* transformed with GFP under T7 promoter and a lac operator
How will you measure the function of your system? Measure GFP output of the cells via flow cytometry. Alternatively, use enzymatic reporter, like luciferase, and measure bulk output of the enzyme.
Artificial cells translate chemical signals for E. coli. (a) In the absence of artificial cells (circles), E. coli (oblong) cannot sense theophylline. (b) Artificial cells can be engineered to detect theophylline and in response release IPTG, a chemical signal that induces a response in E. coli.
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 ā Architecture, Textiles/Fashion, or Robotics ā 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.
My concept is a smart textile integrated with freeze-dried cell-free sensors that changes color and activates neutralizing enzymes when it detects toxic air pollutants in urban environments.
How will the idea work, in more detail? Write 3-4 sentences or more.
The fabric is manufactured by embedding freeze-dried cell-free extracts into the fibers using a specialized coating or encapsulation method. When the wearer enters an area with high concentrations of a specific pollutant, the chemical acts as an inducer that triggers the cell-free genetic circuit. This reaction produces both a chromoprotein for a visible color change and a functional enzyme that actively breaks down the toxin on the surface of the fabric. By using a paper-like matrix within the textile, the biological machinery stays localized and ready to react the moment it comes into contact with the air.
What societal challenge or market need will this address?
This addresses the growing global crisis of air pollution and its impact on public health, especially in hyper-urbanized cities. It provides citizens with a wearable, real-time diagnostic tool that not only alerts them to invisible dangers but also offers a first line of active protection by degrading harmful chemicals around them.
How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?
To handle the one-time use limitation, the textile could be designed with replaceable “bio-cartridges” or patches that are swapped out after an activation event occurs. We can address the water requirement by using the natural humidity in the air or the wearer’s perspiration to provide the initial hydration needed to restart the freeze-dried machinery. For stability, the cell-free components would be encapsulated in protective polymers to prevent degradation from UV light or temperature swings before the sensor is actually triggered.
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 BioBitsĀ® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCRĀ® 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)
Astronauts on long-duration missions face severe nutritional deficiencies because vitamins degrade quickly in space radiation. Vitamin B12 is crucial for neurological health and red blood cell production, yet we currently lack a way to test its levels without sending samples back to Earth. Solving this is vital for deep-space exploration, as it ensures crew health during missions to Mars where resupply is impossible. This is scientifically interesting because it explores how to maintain human homeostasis in an extreme environment using portable synthetic biology tools.
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)
My target is a B12-responsive riboswitch DNA sequence that regulates the expression of a fluorescent protein within the BioBitsĀ® cell-free system.
Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)
The molecular target acts as a biological sensor that detects the presence of active Vitamin B12 molecules. In space, maintaining specific nutrient levels is a constant battle against radiation-induced degradation and physiological changes. By integrating a B12-sensing riboswitch into a cell-free reaction, we can turn a complex nutritional assay into a simple visual test. This allows the crew to monitor their own health and the stability of their food supplies in real-time using minimal equipment, which is essential for surviving the constraints of microgravity.
Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)
My goal is to demonstrate that a BioBitsĀ® cell-free system can accurately quantify Vitamin B12 concentrations in a microgravity environment using fluorescence as a readout. I hypothesize that the B12-responsive riboswitch will remain functional in space and will effectively block or allow the translation of a fluorescent reporter in direct proportion to the vitamin levels present in the sample. The reasoning is that cell-free systems are highly stable when freeze-dried and avoid the complications of maintaining living cultures in orbit. If successful, this provides a low-cost, shelf-stable diagnostic platform that can be adapted to detect many different essential nutrients or even environmental toxins on the International Space Station.
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 need to test astronaut serum samples or rehydrated food extracts mixed with the BioBitsĀ® B12-sensor pellets. I will incubate the samples in the miniPCRĀ® thermal cycler at 37Ā°C to activate the cell-free reaction. I plan to use three controls: a positive control with a known B12 concentration, a negative control with nuclease-free water, and a non-responsive fluorescent DNA template. I will collect data by observing the reaction tubes in the P51 Molecular Fluorescence Viewer to measure light intensity, which correlates to the B12 concentration in the tested samples.
Homework Part B: Individual Final Project
Documentation on my final project page.
We’d like students to start exploring their final project in depth this week! Of your three Aims, for this week you should have at least Aim 1 decided and written down.
Put your chosen final project slide in the appropriate slide deck following the instructions on slide 1:
First Twist order deadline for MIT/Harvard/Wellesley students is Friday, April 3 at 11PM ET
First Twist order deadline for Committed Listeners is Friday, April 10 at 11PM ET. (Your Node Lead will place the Twist order, so please work with them to finalize your constructs and ordering decisions.)
