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
Homework question from Ally Huang
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.