Week 9 HW: Cell-Free Systems

1. 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 first advantage of cell-free protein synthesis (CFPS) over traditional in vivo methods is that it avoids the ethical concerns associated with modifying living cells; CFPS uses cellular machinery such as ribosomes and enzymes. The second advantage of CFPS is the time and cost. CFPS takes around 1-2 hours while cell-based expressions, such as in E. coli, take around a few days to a week, depending on the expression. Third, there is increased biosafety and controllability with CFPS. CFPS are more controllable and programmable. Unlike using living cells which can escape into the environment and potentially cause harm depending on the organism. Lastly

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

A cell-free expression system contains the molecular machinery for transcription and translation containing ribosomes to build the target protein, tRNA to bring amino acids to the target site, RNA polymerase to transcribe DNA into RNA, and translation factors to elongate a DNA template to encode the target protein. The DNA template, usually a plasmid or linear DNA, instructs the cell-free system to produce the protein. The template includes a promoter, a coding sequence, and a terminator. All 20 amino acids are also included. The energy system of the cell, such as ATP to power transcription, and GTP to power translation. Finally and most importantly, the cell-free system’s “environment” is made up of Mg²⁺, K⁺ to stabilise ribosomes and enzymes, NTPs (ATP, CTP, GTP, UTP), for mRNA synthesis, and buffers to maintain pH level, for the reactions to occur efficiently and successfully.

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

To optimise a membrane protein in cell-free expression, I would first choose the lysate based on the protein type: E. coli extract for a bacterial target, and wheat germ or another eukaryotic extract for an eukaryotic target. I would then screen several membrane mimics directly in the reaction—such as mild detergents, liposomes, and nanodiscs—to promote cotranslational insertion and reduce aggregation. I would test multiple conditions for magnesium, temperature, DNA concentration, and reaction format, then compare total expression, soluble fraction, and functional activity rather than yield alone. The major challenges are aggregation, misfolding, poor membrane insertion, and low functional recovery, and these are addressed by matching the CFPS chassis to the target, providing an appropriate lipid environment during synthesis, and screening conditions systematically.

4. 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.

The first reason for a low yield in the target protein is an imbalance in the composition as well as a lack of ATP and GTP. Cell-free systems rely on ATP, GTP, amino acids, salts, cofactors, and an appropriate magnesium and potassium balance. To troubleshoot, one uses the correct measuring tools and is exact in optimising each component especially magnessium and template concentration. One must also ensure the correct temperatures are used with measured time. One can also switch energy usages as cell free systems rely on massive amounts of ATP and GTP. One could use Creatine Phosphate or Glucose.

The second reason for a low yield is inefficient folding and aggregation. In a cell-free extract, proteins can become stuck together, forming inclusion bodies rather than functional proteins. This could be due to a higher concetration in protein. To fix this, one could supplement the mix with purified DnaK/J-GrpE or GroEL/ES complexes to assist with folding. Dropping the incubation temperature can also help. Slower translation often helps the protein find its native conformation.

The final reason for a low yield of the target protein in template degradation is also commonly known as the RNase problem. Cell-free extracts contain endogenous nucleases. DNA template or newly transcribed mRNA is digested before ribosomes can finish their run, and yield drops completely in the first 30-60 seconds, even if the system has enough energy. One can supplement the reaction with 0.5–1.0 U/μL of a recombinant RNase inhibitor. There are also methods to protect linear DNA ends from degradation by the RecBCD exonuclease, such as adding the GamS protein to PCR products. One should also ensure there is no contamination in the tools to minimise surface-bound nuclease contamination.

Homework question from Kate Adamala

Design an example of a useful synthetic minimal cell as follows:

1. Pick a function and describe it.

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

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

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

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

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

a. What would be the membrane made of?

b. What would you encapsulate inside? Enzymes, small molecules.

c. 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)

d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

3. Experimental details

a. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)

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