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.

Comparative to traditional methods, cell free systems:

• Permit the incorporation of non-canonical amino acids (NCAA’s) for extended protein functionality not observed in nature.
• Permit the synthesis and collection of difficult to express proteins, such as trans-membrane proteins.
• Allow the immediate use of linear fragments in solution. This offers high flexibility for projects involving prototyping at frequent intervals; no need for plasmid construction and subsequent cloning.
• Permit greater control of matrix composition during biochemical processes. Proceed with crude extracts for high-throughput, inexpensive studies, or calibrate its composition (such as in PURE) to examine or promote specific processes.

Cell-free is uniquely superior for use in environments which do not support cell production techniques, such as in materials! i.e a cell-free system may be inactivated by lyophilization then subsequently reactivated upon rehydration.

Describe the main components of a cell-free expression system and explain the role of each component.

• Engineered sequences for translation; typically plasmids, but also possible with linear fragments. The core information for what is to be expressed.
• Cellular lysate containing required molecular machinery for Tx/Tl (ribosomes, RNA polymerase etc). Permits the expression of the engineered sequences.
• Source of energy-rich compounds and nucleosides. Provide free energy for enzymes in the reaction to do work, as well as necessary substrates for building mRNA.
• Buffering compounds: stabilise reactions and maintain consistent pH.

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.

In living cells, energy regeneration is typically mediated by sequential chains of catabolic and anabolic reactions, by which the cell will sustain protein-synthesis activities; these pathways can be absent in cell-free systems. Phosphoenolpyruvate (PEP) is commonly used to regenerate ADP molecules into ATP, providing a ‘continuous’ supply with the caveat that its concentration must be ample for the entire duration of the intended experiment. Glucose-6-phosphate (G6P) is a promising, affordable alternative to PEP which still functions as a reducing agent.

Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

Each approach presents its own distinct advantages and limitations, often dependent on the specific source for which the expression system is based. While prokaryotic permits high protein yields, simplicity in genetic engineering, and the ability to synthesise in extreme conditions (Archeal extremophile extracts), post-translational modifications are limited and only chaperones native to prokaryotic species are available. Eukaryotic extracts are diverse, permit fast lysate preparation, and mirror mammalian systems, yet often are also subject to high-cultivation costs and low protein yields.

Prokaryotic: Cas12a, modified via the inclusion of NCAA’s to extend its trans-cleavage properties. Useful when expressed in high yields as the main component of a signal-amplifying biosensor for.

Eukaryotic: any protein intended for use in the human body! Likely to produce proteins with higher-quality folding, as well as overall be more compatible with human biochemistry.

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.

Membrane proteins are often flexible and structurally unstable, and are typically expressed and purified from live-culture systems in which they are first correctly folded and inserted into the cellular membrane by a specific enzyme. To mirror this process and extend the stability of synthesized membrane proteins during storage, one such approach could be to encourage their integration with artificial vesicles. Whilst this technique may improve their stability, it could prove as a hindrance when further purification is required to ‘detach’ the membrane proteins and reintegrate them into the host organism.

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.

Insufficient energy source: recalibrate concentration. Incompatible lysate: identify suitable alternative. Inefficient codon usage: reoptimise codons for expression in specific lysate.

Homework question from Kate Adamala

Pick a function and describe it.

1. What would your synthetic cell do? What is the input and what is the output?

Produce and excrete magnetic inclusions (magnetosomes).

Input: Isopropyl β-D-1-thiogalactopyranoside (IPTG).
Output: Magnetite vesicles.

2. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

No, membrane encapsulation is required for magnetite vesicle formation.

3. Could this function be realized by genetically modified natural cell?

Yes! However, likely not at a scale and yield which is industrially significant.

4. Describe the desired outcome of your synthetic cell operation.

Minimal cells are cultivated inside a bioreactor, with capability to support the production of various valuable compounds in response to specific signalling molecules. When IPTG is added to the system, culture responds by producing and secreting magnetite vesicles.

Design all components that would need to be part of your synthetic cell.

1. What would the membrane be made of?

Unsaturated fatty acid bilayer to enhance fluidity and facilitate vesicle formation.

2. What would you encapsulate inside?

Proteins associated with the MamAB gene cluster would constitute the most significant inclusions, specifically MamE (magnetite and vesicle maturation), MamI + MamL (magnetosome membrane formation).

3. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason?

Prokaryotic is preferred; Tx/Tl system can primarily be derived from the magnetotactic model organism AMB-1.

4. How will your synthetic cell communicate with the environment?

IPTG is membrane permeable. Magnetite vesicles may be freely transported via budding and shedding.

Experimental details

1. List all lipids and genes.

mamAB gene cluster: magnetite vesicle formation.
LacI repressor: receptor for IPTG.
IPTG ligand: ligand for LacL deactivation.

2. How will you measure the function of your system?

Due to the conductive nature of magnetite, I would use electrochemical conductometry to monitor the protein yield in real time in response to the presence of IPTG.

Homework question from Peter Nguyen

Write a one-sentence summary pitch sentence describing your concept.

I propose a biosensor embedded in a reusable, broach-like microfluidic capable of the detection and artistic indication of a broad-spectrum of aerosolised compounds.

How will the idea work, in more detail?

The seemingly inescapable routine demanded by contemporary working culture is often the culprit for distorting the passage of time, accelerating our perceptions to feel as if months pass by frictionless, leaving nothing to hold but vague recollections of mundanity. What if we had a unique anchor to distinctly summarise each day?

The broach would be composed of photoablated acrylic disk sandwiched by disposable semi-permeable membranes and a fixed, chemicapacitive backing. As the user goes about their day, aerosols and airborne particulates attach and diffuse passively through the external membrane into the grid-like, photoablated, aqueous buffer beneath. The cell-free system inhabiting the aqueous buffer would be composed primarily of constructs engineered to transcribe proteins of varied charge, each controlled by a unique transcription activator-like effector (TALE) repressor which interacts with a specific aerosol substrate. Conformational binding would detach the repressor and permit localised protein expression, corresponding to a measurable increase in the net charge of the pore via a measurable change in capacitance at the pore-chemicapacitive backing interface. Capacitance values retrieved across the porous grid would then be transformed and represented digitally (i.e hex code, greyscale value, 3D peaks / troughs) to create a one-off abstract representation of the day experienced according to the unique air composition. To reset the device, the disposable membranes would be replaced, and the pores of the broach flushed and refilled with the cell-free system.

What societal challenge or market need will this address?

Routine, and the departure from spontaneity, is inevitable as we age and mature. Although a minor combatant, the concept aims to help its user, and broader communities as a whole, integrate a consistent element of novelty into their daily lives as a means to provide a substrate for the formation of episodic memories in an otherwise routine day. It is hoped that observation and reflection upon this small, consistently inconsistent visualisation of daily difference will help its user slow down momentarily and appreciate even the most seemingly mundane of days.

How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

The versatility of capacitance-based sensors permits implementation of a wide range of perpetually reusable configurations, with the simplest requiring just two conductive plates and a thin dielectric layer. Flushing and refilling the device presents the greatest challenge; how might residual reagents skew capacitive readout? How might the fresh aqueous cell-free circuit be suitably stored and supplied to the user as to permit daily refill? Where may a spent cell-free solution be safely disposed of? For this prompt, I envision a world where the production of constructs and enzymatic intermediaries is possible at-home via the democratisation of processes (and associated reagents) such as oligonucleotide synthesis and gibson assembly, by which specialised ‘one-time’ cell-free circuits may be built and safely deployed.

Homework question from Ally Huang

Provide background information that describes the space biology question or challenge you propose to address.

Energy preservation is of particular importance when considering deep space exploration. Isolation, and the inability to receive prompt resupply, place onus upon the use of highly self-sufficient energy systems such as nuclear. Radioisotope thermoelectric generators (RTG) are a prime example, powering and permitting the incredible endurance of spacecraft such as the Voyager probes. Nuclear systems, such as NASA’s SR-1 fission-powered spacecraft Freedom, are likely to play an increasing role within our presence in space.

While nuclear energy sources such as RTG’s are typically designed with strong failsafes and redundancies, their critical failure and spillage could prove catastrophic for resource-limited planetary colonies, threatening the habitability of already challenging environments.

Bio-remediation presents a possible solution to the stabilisation of environments intended to support life post-incident. My proposal seeks to identify and refine a cell-free system (or otherwise!) as a bio-remediation tool to stabilise irradiated environments in space.

Name the molecular or genetic target that you propose to study.

Bacillus sphaericus is a radioresistant prokaryotic bacterial species featuring an outermost paracrystalline S-layer that is described to possess radioabsorptive properties; effectively a bacterial mop! My proposal would investigate the pairing of the pprA radio-responsive promoter from Deinococcus radiodurans with the S-layer encoding sbpA gene from B. sphaericus, in a single synthetic construct.

Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses.

sbpA proteins are capable of self-assembling to form organised S-layer lattice structures; this level of autonomy is highly advantageous for resource-limited settings in space. Confirming sbpA expression and consequent S-layer formation in a cell-free system would be pivotal in scaling the process to the extent required for utility as a feasible radioactive cleanup method.

Clearly state your hypothesis or research goal and explain the reasoning behind it.

The concentration of sodium bicarbonate-¹³C in solution will be inversely proportional to sbpA expression post-filtration.

The objective is to examine the efficacy of sbpA two-dimensional lattice structures in temporarily sequestering bicarbonate-¹³C present in an aqueous solution. Stable and non-toxic, Carbon-13-labled species presents a feasible analog to hazardous radionuclides for investigating the aptitude of synthetic S-layers in-solution for nuclear cleanup. If we are able to observe a decrease in the concentration of the tracer after nucleopore filtration, we can likely identify causality between S-layer assembly and isotope entrapment.

Outline your experimental plan.

Two 1.5 ml reaction tubes:

Tube A: BioBits® pellets + sodium bicarbonate-¹³C

Tube B: BioBits® pellets + sodium bicarbonate-¹³C + sbdA constructs

1. sbpA gene codon-optimised for expression with BioBits® cell-free system. sbpA sequence flanked in-silco with T7 promoter + RBS (5’) and T7 terminator (3’) and primers incorporated. Cloned via miniPCR® thermal cycler. Incorperate with 80uL nuclease-free liquid.

2. Pipette 15 μl of cloned sbpA DNA into reaction tube B, incubate at 30c for 4 hours.

3. Pipette 1.0 mL of 8.5 µg sodium bicarbonate-¹³C stock into tube A and B. Stow in a shaker-incubator at 25c for 4 hours.

4. Filter A and B through nucleopore membrane filter; evaluate entrapment via Isotope Ratio Mass Spectrometry (IRMS).

References

Calhoun, K.A. and Swartz, J.R. (2005). Energizing cell‐free protein synthesis with glucose metabolism. Biotechnology and Bioengineering, [online] 90(5), pp.606–613. doi:10.1002/bit.20449.

Carpenter, E.P., Beis, K., Cameron, A.D. and Iwata, S. (2008). Overcoming the challenges of membrane protein crystallography. Current Opinion in Structural Biology, [online] 18(5), pp.581–586. doi:10.1016/j.sbi.2008.07.001.

Murat, D., Quinlan, A., Vali, H. and Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. Proceedings of the National Academy of Sciences, [online] 107(12), pp.5593–5598. doi:10.1073/pnas.0914439107.

Paul, N.L., Carpa, R., Ionescu, R.E. and Popa, C.O. (2025). The Biomedical Limitations of Magnetic Nanoparticles and a Biocompatible Alternative in the Form of Magnetotactic Bacteria. Journal of Functional Biomaterials, [online] 16(7), p.231. doi:10.3390/jfb16070231.

Pui Yan Wong, Mal, J., Sandak, A., Luo, L., Jian, J. and Pradhan, N. (2024). Advances in microbial self-healing concrete: A critical review of mechanisms, developments, and future directions. The Science of The Total Environment, 947, pp.174553–174553. doi:10.1016/j.scitotenv.2024.174553.

Zemella, A., Thoring, L., Hoffmeister, C. and Kubick, S. (2015). Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. ChemBioChem, [online] 16(17), pp.2420–2431. doi:10.1002/cbic.201500340.