Week 9 — Cell-Free Systems

General homework questions

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.

Cell-free protein synthesis is a biochemical technique that uses the cellular transcription and translation machinery extracted from cells (lysates) and operated in a test tube. Its principal advantage is that it bypasses the cellular barriers and restrictions that come with maintaining cell homeostasis, resulting in an open, flexible and controllable system.

The most significant benefits compared to traditional in vivo methods are:

• High sample throughput
• Insensitivity to initial conditions - scanning the sample provides a faithful description of its properties throughout
• No need for special expertise, with sample preparation largely automated
• Objective results free from multiple human assays

1. Flexibility and Direct Access to the Reaction Environment Cell-free expression systems provide an open channel for direct intervention at the reaction site. Real-time monitoring and adjustments: You can take samples, measure reaction kinetics and adjust chemical parameters. Control of physicochemical conditions: Allows direct modification of pH, ion concentration (e.g. (Mg^{2+})) and redox potential to optimize protein folding.

2. Control of Experimental VariablesElimination of parasitic metabolism: All the energy of the system can be channeled exclusively to the synthesis of the protein of interest, with no risk of the host cell degrading the protein through its own enzymes (proteases). Linear template-based synthesis: Reactions can directly use linear DNA fragments (such as PCR products), eliminating the need for prior cloning into plasmids, which speeds up the process from a few weeks to a few hours.

3. Production of “Toxic” or Troublesome Proteins Cell Death: Due to their antimicrobial properties, many useful proteins would be toxic to each cell which produces them, keeping them from expressing the molecules. The absence of living cells in cell-free systems allows the expression of such proteins, as they will not suffer from the consequences like the living cells. Lipidic environment: By adding detergents or lipids, such as liposomes or nanodiscs, hydrophobic proteins can be placed in an environment similar to cellular membranes, thus allowing for their direct synthesis into these membranes.

Cell-free synthesis is better than cellular synthesis:

Toxic proteins: Cells that produce proteins that are toxic to their membranes or interfere with their metabolism simply cease to live. However, cell-free systems have no membranes.

Incorporating the extended genetic code: it is much simpler and more efficient to place unnatural amino acids or fluorescent/isotopic labels into cell-free systems: there is no need to worry about the competition of natural amino acids, as there is no cell membrane to block entry of proteins or metabolites.

Speed and rapid prototyping: There is no need for long cloning and cell cultivation steps (hours vs. days). All the energy of the system is focused solely on the protein of interest.

Complex and unconventional metabolic processes: Systems can withstand a wider range of temperatures and pH and are not limited by the homeostasis and toxicity of cells

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

In a cell-free system, a cell is extracted from a living organism and its contents are used. The main components of such a system and their role are described below:

1. Cell Lysis: In the cell-free system, the cells are broken down by the process of cell lysis. During lysis, the cell membrane is disrupted,

2. Cellular Extract

• This is the heart of the system. In this fraction, the machineries that carry out transcription (generation of RNA) and translation (production of proteins from RNA) reside.

• It is obtained, typically, from bacteria (E. coli), wheat germ or rabbit reticulocytes.

• The cellular extract is constituted by ribosomes, tRNA, aminoacyl-tRNA synthetases, translation factors (initiation, elongation, termination) and molecular chaperones to fold and stabilize translated proteins

2. Nucleic Acid Matrix (Genetic Information) It is the instruction on the basis of which the desired protein will be built. DNA or RNA: The system can be initiated using a DNA molecule (plasmids or linear fragments) that is first transcribed into mRNA, or directly with pre-synthesized mRNA.

3. Energy Regeneration System

Because of the energetic demands of protein synthesis at the molecular level, we need to provide energy packets in the form of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to the ribosomes all along the way.

Components: Phosphate donors (e.g. phosphoenolpyruvate or creatine phosphate) and specific enzymes (e.g. kinases)

4. Building Blocks (Monomers) Amino Acids: They are used for synthesizing the polypeptide chain. They mix all 20 essential amino acids.

Nucleotides (dNTPs / NTPs): They are the building blocks used for transcribing DNA to mRNA or for replicating the viral genome in some complex systems.

5. Cofactors and Salts: Metal Ions: These are ions like magnesium ((Mg^{2+})) and potassium ((K^{+})) that stabilize ribosomes and facilitate the binding of tRNA to mRNA. Buffer: This maintains the optimal pH to prevent protein denaturation.
6. Special Options (optional use in specific cases)
   • RNA Polymerase (optional): e.g. T7 phage enzymes for fast transcribing.
   • Molecular Crowding Agents (optional): for e.g. PEG to increase the density of the solution and mimic the living cell to get assembled large structures like virus.
   • Artificial Membranes (optional). For e.g. liposomes for synthesis of membrane or creation of artificial cells.

3 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.

One of the major challenges in designing cell-free protein expression is to regenerate the energy-storing molecules ATP and GTP. The microsome system is particularly inefficient in permitting them to regenerate because of high rates of translation and transcription, which consume enormous amounts of ATP and GTP. The production comes to a halt within minutes as energy supplies become exhausted, leading to accumulation of inorganic phosphate and exhaustion of cofactors that lead to inhibition of enzymes.

There are several cell-free systems in which the cell’s ability to synthesize ATP has been lost through isolation or genetic modification. One of the most widely used and highly effective means of providing a steady source of ATP in these cell-free systems is through the use of a high-energy bond donor, creatine phosphate (CrP), in combination with the enzyme creatine kinase (CrK).

Details:

• ATP recharge: When ATP is used by cellular machinery to synthesize proteins, it is converted into adenosine diphosphate (ADP).

• Role of the enzyme: The enzyme CrK catalyzes the transfer of a phosphate group from the CrP molecule directly to the ADP molecule.

• Continuous cycle: This transfer regenerates ATP, which again becomes available to support protein synthesis.

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

  The use of prokaryotic (bacteria such as E. coli) and eukaryotic (yeast, mammalian cells) systems in the laboratory differs radically in terms of speed, cost, complexity of manipulation, and ability to correctly fold complex proteins. The prokaryotic systems have a very high rate of growth (doubling time 20-30 min), low costs, the protein foldingis simple; often forms inclusion bodies (requires denaturation/renaturation), has excellent Scalability (large-scale fermentation, easy to automate), and it’s main applications are production of simple recombinant proteins, plasmids, insulin. The eukaryotic sistems are slow in growth, high in costs, protein folding is advanced; correct folding, capable of post-translational modifications (glycosylation), the scalability is more difficult, requires special bioreactors and fine monitoring,it’s main applications are monoclonal antibodies, complex vaccines, human genetics studies

  5 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.

  Optimized design of in vitro experiments for membrane proteins relies on combined synthetic biology approaches, statistical methods (DoE) and artificial environment simulations to overcome the instability of these proteins and their difficulties in lipid integration. To optimize folding and integration efficiency, researchers use DoE methodologies (such as Fractional Factorial Design) to simultaneously test multiple variables in the test tube:Lipid Composition: The ratio of phospholipids (e.g. DOPC, DOPE, DOPS) to sterols (cholesterol).Nanodiscs and Polymers: The use of amphipathic polymers (known as SMA - Styrene Maleic Acid) to create nanodiscs that stabilize the native protein without the need for harsh detergents.Additives: The concentration of metal ions, reducing agents (DTT), and molecular chaperones added to the translation mix.

  6 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.

Low yield in cell-free systems (CFPS) is usually caused by premature template degradation, energy depletion, or protein misfolding. Optimization requires a sequential approach to DNA template, reaction parameters, and extract integrity. Identify the root cause of low yield and apply appropriate solutions:

1. DNA/mRNA template issues Cause: Nucleases in the mixture or contaminants in purification kits (salts, ethanol, solvents). Strategy: Purify DNA with dedicated spin column kits and avoid extraction by agarose gel elution. Always use RNase inhibitors. Cause: Rigid mRNA secondary structures at the ribosome binding site (RBS) or rare codons that block translation. Strategy: Optimize the codon sequence for the host organism (e.g. E. coli). You can use a dedicated tool, such as the IDT Codon Optimization Tool, to adjust the sequence. Cause: Inadequate DNA concentration (too low or too high). Strategy: Test DNA concentrations ranging from 10 ng to 250 ng per 50 (\mu L) reaction to balance transcription and translation levels.
2. Reaction conditions and energyCause: Rapid consumption of high-energy molecules (ATP, GTP), leading to the cessation of synthesis.Strategy: Add an energy regeneration system (such as creatine phosphate/creatine kinase or phosphoenolpyruvate) and consider continuous exchange reactions (cecf) to increase the lifetime of the system.Cause: Too high reaction rate at standard temperatures (e.g. 37°C) which prevents correct protein folding.Strategy: Reduce the incubation temperature (between 25°C and 30°C) and extend the reaction time to slow down the process and allow the protein to adopt the correct conformation.
3. Protein degradation and solubility issuesCause: Degradation of the target protein by endogenous proteases present in the cell extract.Strategy: Supplement the reaction with a mixture of protease inhibitors to protect the protein.Cause: Complex proteins are synthesized as insoluble inclusions or require cofactors/chaperones.Strategy: Add molecular chaperones directly to the reaction mixture or use additives for disulfide bond folding, depending on the characteristics of the protein.Cause: Purification tags (e.g. His-tag) alter the native structure of the mRNA.Strategy: Change the position of the tag (N-terminal vs. C-terminal) or test a different solubility tag.

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?

The proposed function is a synthetic sensor–actuator cell: the input is a chemical signal associated with the intestinal environment or the presence of a bacterial target, and the output is an antibacterial bacteriocin that inhibits Clostridium. In a thesis formulation, you can say that the synthetic cell “senses” a molecular cue and then produces a local antimicrobial response, with the aim of limiting the growth of clostridia. The desired outcome is that, in the presence of the input signal, the synthetic compartment produces and releases an active bacteriocin, which in turn reduces the growth of Clostridium from the external environment. For a theme, the most elegant choice is to use a small, well-characterized bacteriocin, such as pediocin PA-1, as the literature shows both its heterologous production in E. coli and relevant antibacterial activity. Cell components The membrane can be described as a lipid bilayer of phospholipid + cholesterol, which is standard in many liposome-based synthetic cell models. Inside I would encapsulate a bacterial-like cell-free expression system, plus the DNA of the genetic circuit encoding the chosen bacteriocin and, if you want controlled export, a liposome-compatible membrane protein channel/pore

b.Could this function be realized by cell-free Tx/Tl alone, without encapsulation? The desired outcome is that, in the presence of the input signal, the synthetic compartment produces and releases an active bacteriocin, which in turn reduces the growth of Clostridium from the external environment.

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

For a theme, the most elegant choice is to use a small, well-characterized bacteriocin, such as pediocin PA-1, as the literature shows both its heterologous production in E. coli and relevant antibacterial activity d.Describe the desired outcome of your synthetic cell operation.

The desired outcome is that, in the presence of the input signal, the synthetic compartment produces and releases an active bacteriocin, which reduces the growth of Clostridium in the external environment. For a theme, the most elegant choice is to use a small, well-characterized bacteriocin, such as pediocin PA-1, as the literature shows both its heterologous production in E. coli and relevant antibacterial activity.

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

a.What would be the membrane made of?

The membrane can be described as a lipid bilayer of phospholipid + cholesterol, which is standard in many liposome-based synthetic cell models.

b.What would you encapsulate inside? Enzymes, small molecules. Inside I would encapsulate a bacterial-like cell-free expression system, plus the DNA of the genetic circuit encoding the chosen bacteriocin and, if you want controlled export, a liposome-compatible protein membrane channel/pore.

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)

The Tx/Tl system should be bacterial, since the chosen bacteriocins come from lactic acid bacteria and the circuit can be described as a minimalist bacterial module. Communication with the environment is done by the liposomal membrane allowing or regulating the passage of small molecules, and the final product diffuses to the target bacteria.

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

The input signal must be a molecule small enough to diffuse through the membrane or through a simple channel. The output is the bacteriocin, which must reach the outside by diffusion or controlled membrane permeabilization.

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.)

A. Simple Lipids (Glycerides and Waxes) They are esters of fatty acids with various alcohols: Triglycerides: The main form of energy storage (fats from food and adipose tissue). Waxes: Esters of fatty acids with higher alcohols with high molecular weight (e.g. lanolin, beeswax).

B. Complex Lipids (Phospholipids and Glycolipids)They also contain other elements (phosphorus, sugars) and have a structural role in cell membranes: Phospholipids: They form the lipid bilayer of cell membranes. Examples: lecithin, cephalin.Glycolipids: They contain carbohydrates; they are found especially in nervous tissue and the brain (e.g. cerebrosides, gangliosides). Sphingolipids: Essential components of the membrane of nerve cells (e.g. myelin).

C. Lipid Derivatives and Precursors Sterols: Polycyclic structures, the best known being cholesterol (precursor for hormones and vitamin D). Fatty acids: Free or esterified (e.g. Omega-3, Omega-6). Other lipids: Fat-soluble vitamins (A, D, E, K), steroid hormones (cortisol, testosterone) and prostaglandins.

b.How will you measure the function of your system? Functionality can be measured by a reporter, such as GFP or luciferase, to verify expression. The final effect is measured by reducing Clostridium growth.

Homework question from Peter Nguyen

We can use cell free systems to introduce sensors into clothing making travelling in dangerous countries safer. For example creating a swab that changes colours when detecting proteins present in deadly bacterias like Clostridium botulinum or Listeria monocitogenes. The fluorescent shift could be detected by the phone camera, and when visiting less developed contitents, we could use these swabs before eating.

Homework question from Ally Huang

There are many factors that increase the probability of a human to become ill in space. The constant stress on the body causes cortisol to rise which inhibits t cells activity, microgravity increases the floating time of pathogens in already isolated chambers where air and resources are constantly recirculated, while also helping the bacteria form stronger biofilms. Because of this many astronauts suffer the reactivation of pre existent dormant infections like herpes while also contacting respiratory, urinary and gastro-intestinal diseases. While cultivating bacterias is efficient in protein synthesis it also requires special conditions and equipment like special freezers and are falling behind, especially in longer journeys. That’s why free-cell systems can be used to detect the presence of these antigens in people and the water supplies while also storing proteins capable of fighting them. As E.coli is one of the most common bacterias in utis we can use discs to detect it’s antigens and prospective antibiotics.