Week 9 HW: Cell Free Systems

Part 1: General Homework Questions

1. Cell’s survival is the priority. In CFPS, the “cell” is broken open, leaving only the machinery. The advantages include more direct access (adding non-canonical AA, detergents, chaperones) or an open system to monitor the reaction in real-time and adjust variables. One case where this is more beneificial would be in toxic proteins; if the protein kills a living host, it can still be produced in a cell-free system because there’s no “life” to extinguish. Another case could be for rapid prototyping, where CFPS allows for cycles in hours rather than the days required for usual cell transformation and growth.
2. Major components: crude extract provides things like ribosomes, RNA polymerase, translation factors, the DNA template encodes the protein, the energy mix provides energy, amino acids provide building blocks for proteins and cofactors/salts provide ions that help stability and enzyme activity.
3. Energy provision regeneration is critical because translation is energy-intensive. Every peptide bond requires the hydrolysis of multiple high-energy phosphate bonds. Without regeneration, ATP levels plummet in minutes, and the accumulation of inorganic phosphate inhibits the reaction. Something possible for continuous supply would be to use Secondary Energy Source, such as the Creatine Phosphate/Creatine Kinase system. Creatine kinase transfers a phosphate group from creatine phosphate back to ADP, maintaining a steady-state concentration of ATP throughout the batch reaction.
4. Prokaryotic: High yield, fast and cheap. A protein that might be produced is GFP, that is simple and doesn’t require complex folding or glycolysations. Eukaryotic systems provide lower yield but are capable of complex post-translational modifications like glycosylation and proper disulfide bond formation. A protein that might be produced in one of these systems is human insulin, which requires specific folding and bridges that the eukaryotic machinery handles better.
5. When encountering low protein yields in a cell-free system, the first step is often to investigate template stability. In many extracts, endogenous nucleases are present that can rapidly degrade linear DNA templates. A common troubleshooting strategy is to switch to a circular plasmid or supplement the reaction with nuclease inhibitors like Gam protein. A second common culprit is codon bias, where the genetic sequence of the target protein utilizes codons that are rare within the organism from which the extract was derived. This can be addressed through synonymous gene sequence optimization or by using specialized extracts supplemented with rare-target tRNAs. Finally, the concentration of magnesium ions is a critical variable that often requires a titration experiment. Because magnesium is essential for ribosome assembly but inhibitory at high concentrations, performing a series of reactions across a gradient is a standard strategy to find the “sweet spot” for a specific proteins.

Part 2: Homework Questions from Kate Adamala

Designing a cell-free experiment for membrane proteins introduces the unique challenge of hydrophobicity, as these proteins often aggregate or misfold without a lipid environment. To address this, one can incorporate synthetic surfactants or detergents into the reaction to keep the protein soluble during synthesis. Alternatively, a more biomimetic approach involves adding nanodiscs or liposomes directly into the cell-free mix, providing a membrane-like scaffold for the protein to insert into co-translationally. The primary advantage here is that the open nature of the system allows you to precisely control the lipid composition to optimize the stability and activity of the membrane protein without the toxicity issues often seen in living hosts.

The structural components of this synthetic cell would include a liposome membrane composed of POPC and Cholesterol to ensure durability and prevent leakage. Inside, we would encapsulate an E. coli S30 extract to provide the metabolic hardware, along with an energy regeneration system like phosphoenolpyruvate (PEP) and the necessary amino acids. A bacterial Tx/Tl system is ideal here because it is compatible with well-characterized, small-molecule-modulated promoters. To allow communication with the environment, the cell must express a membrane channel; specifically, the GlpF gene (glycerol facilitator), which is naturally permeable to arsenite, would be integrated into the design.

From an experimental standpoint, the “genetic software” inside the liposome would consist of a circuit including the arsR repressor gene, a corresponding Pars promoter, and a reporter gene such as Firefly Luciferase (luc). When arsenic enters through the GlpF channels, it binds to the ArsR protein, releasing it from the DNA and allowing the cell-free machinery to transcribe and translate the luciferase protein. The function of the system is then easily measured using a luminometer or a dark-box camera; the intensity of the light emitted serves as a direct, quantifiable proxy for the concentration of arsenic in the water sample.

Part 3: Homework Questions from Peter Nguyen

I propose “Genetic Glass,” a bio-integrated window coating containing freeze-dried cell-free systems that produce UV-blocking pigments in response to high-intensity solar radiation.

The concept involves incorporating lyophilized cell-free machinery into a transparent, porous polymer film applied to building facades. When the humidity in the air or a specialized hydration channel activates the system, it remains “primed” to sense specific environmental triggers. In this design, a light-sensitive protein initiates the expression of melanin or a high-density chromogenic protein. As the sun hits its peak intensity, the window film darkens or becomes opaque, providing dynamic, biological shading that reduces the building’s solar heat gain without requiring mechanical shutters or electricity.

This addresses the massive energy consumption associated with HVAC systems in glass-heavy urban architecture. Traditional “smart glass” requires complex wiring and rare-earth metals; a cell-free biological coating offers a sustainable, “grown” alternative that can be retrofitted onto existing structures to improve energy efficiency and reduce the carbon footprint of cooling large commercial buildings,

To address the one-time use limitation, the design utilizes a “layered capsule” approach within the polymer matrix. Instead of all reagents activating at once, different micro-pockets are engineered to degrade at different rates or under different osmotic pressures, allowing for multiple “cycles” of pigment production over a season. Furthermore, the use of extremophile-derived extracts can ensure that the transcription-translation machinery remains functional even under the high-heat and high-radiation conditions typical of a building’s exterior.

Part 4: Homework Questions from Ally Huang

As we look toward Moon bases and Mars colonies, growing our own food is non-negotiable, but microgravity and cosmic radiation can trigger “silent” plant diseases that ruin a harvest before symptoms are visible.

In closed-loop life support systems, a single fungal or bacterial outbreak could compromise an entire mission’s food supply. Microgravity alters plant immune responses and can increase the virulence of certain opportunistic pathogens. Detecting these threats early is difficult because traditional visual inspections only catch infections once physical damage is done. Developing a rapid, molecular-based detector for plant pathogens using the BioBits® system would allow astronauts to quarantine infected crops and save the rest of the habitat’s yield.

The target is the 16S rRNA sequence (for bacteria) or ITS (Internal Transcribed Spacer) RNA (for fungi) specific to common greenhouse pathogens like Pseudomonas syringae.

Pathogens shed genetic material into the hydroponic water or onto leaf surfaces long before the plant wilts. By targeting highly conserved but genus-specific RNA sequences, we can identify exactly which “hitchhiker” microbe is multiplying in the space farm. This is scientifically interesting because it explores how microbial “shedding” occurs in microgravity and provides a low-resource way to maintain the delicate microbiome balance required for space-based agriculture.

Hypothesis: A BioBits® cell-free reaction, triggered by a pathogen-specific RNA toehold switch, will provide a visible fluorescent “warning light” in less than 60 minutes when exposed to contaminated plant swabs. Reasoning: In space, we cannot afford the power or mass of a full diagnostic lab. BioBits® is ideal because it is shelf-stable at room temperature and only requires rehydration. By engineering a “toehold switch”—an RNA sensor that only allows a fluorescent protein to be translated when it binds to the pathogen’s RNA—we create a binary (Yes/No) sensor. If the sample contains the pathogen’s genetic “signature,” the BioBits® pellet will glow, providing an immediate, actionable result for the crew.

The experiment will use three sets of BioBits® pellets containing the RNA sensor for P. syringae. Sample 1 (Negative Control): Rehydrated with sterile water. Sample 2 (Positive Control): Rehydrated with a solution containing synthetic target RNA. Sample 3 (Test): Rehydrated with a “leaf wash” from a simulated infected plant. All samples will be incubated in the miniPCR® at $37. After one hour, the tubes will be placed in the P51 Viewer. We will collect qualitative visual data (presence/absence of light) and quantitative data by photographing the tubes and measuring fluorescence intensity.