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. Name at least two cases where cell-free expression is more beneficial than cell production.

The main advantage of cell-free protein synthesis is the complete control we can get over experimental conditions. Unlike in vivo systems, where the cell regulates gene expression, metabolism, and resource allocation, in cell-free systems, the researcher directly defines all components and conditions.

This means that there is flexibility on what you want or not, such as specific DNA, enzymes, cofactors, salts, or inhibitors. There are no cellular constraints, such as toxicity issues. There is no need for cloning, transformation, or cell growth, which makes experimentation faster. There is overall full control over experimental variables like transcription and translation rates, the environment and energy.

Examples where cell-free protein synthesis is more beneficial:

1.- Proteins that would kill or stress a cell can be easily and safely produced. 2.- When there is a need to do a quick prototype to test a genetic circuit without actually needing to grow a cell 3.- In case you want to incorporate non-natural amino acids, it is easier to do it in a cell-free system.

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

• DNA Encodes the target protein

• RNA polymerase Transcribes DNA into mRNA

• Ribosomes Translates mRNA into protein

• tRNAs and amino acids Subparts to synthesize protein

• Energy system Powers transcription and translation. Some examples are ATP, GTP.

• Enzymes and cofactors Provides the necessary biological machinery

• Buffers Maintains optimal reaction conditions/environment.

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.

Protein synthesis consumes large amounts of ATP and GTP. Without regeneration, the reaction stops quickly, and the protein yield is very low. A method to ensure continuous ATP supply would be the Phosphoenolpyruvate (PEP) system, which consists of adding PEP to ADP to obtain ATP.

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

I would produce GFP in the prokaryotic system because its simple, we can make a lot for cheap, and it does not need complex folding or modification.

I would produce antibodies in the eukaryotic system because it requires complex foldings and modifications.

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 difficult to express because they are hydrophobic, require a lipid environment, and tend to misfold or aggregate in aqueous systems.

To design a successful cell-free experiment, it is important to mimic the natural membrane environment. This can be done by adding lipid-based structures such as liposomes or nanodiscs, or by using mild detergents to stabilize the protein during synthesis.

Additionally, experimental conditions should be optimized to improve folding and stability. This includes adjusting temperature (often lower temperatures improve folding), controlling the expression rate to avoid aggregation, and adding molecular chaperones to assist in proper protein folding.

By taking advantage of the flexibility of cell-free systems, the environment can be engineered to support correct membrane protein insertion and function.

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.

One possible reason for low protein yield is energy depletion. Protein synthesis requires large amounts of ATP and GTP, and if these are not regenerated, the reaction will stop early. This can be addressed by including an energy regeneration system such as phosphoenolpyruvate or a glucose-based system.

A second possible cause is non-optimal reaction conditions, such as incorrect magnesium concentration, pH, or salt levels. These factors strongly affect enzyme activity and ribosome function. The solution is to systematically optimize buffer conditions for the specific system being used.

A third reason could be protein misfolding or degradation. Some proteins require specific folding conditions or assistance from chaperones. This issue can be mitigated by lowering the reaction temperature, adding chaperone proteins, or including stabilizing agents in the reaction mixture.

Homework question from Kate Adamala

Example design: synthetic minimal cell that detects doxycycline and activates bacteria

This system is not directly measuring bacterial killing, but rather detecting the presence of an antibiotic and converting it into a readable biological signal.

Function

• A synthetic minimal cell that acts as a chemical translator between the environment and bacteria
• It detects doxycycline and converts that signal into IPTG release
• IPTG then activates GFP expression in nearby engineered bacteria

Input and output

• Input: doxycycline
• Output of synthetic cell: IPTG
• Final system output: GFP fluorescence in bacteria

Could this be done with cell-free Tx/Tl alone?

• No, not properly
• Without encapsulation:
  • IPTG would diffuse freely
  • No controlled release
  • No real “cell-like” behavior
• Encapsulation is necessary to:
  • store the output molecule
  • release it only after activation

Could this be done with a genetically modified natural cell?

• Yes, it could be engineered directly in bacteria
• However, synthetic minimal cells offer:
  • modular design
  • easier swapping of sensing modules
  • no need to maintain living cell viability
• Synthetic and natural systems can work together

Desired outcome

• Without doxycycline:

  • no IPTG release
  • bacteria remain non-fluorescent

• With doxycycline:

  • synthetic cell activates
  • IPTG is released
  • bacteria express GFP

Components of the synthetic minimal cell

• Lipid vesicle membrane
• Cell-free transcription/translation system
• DNA encoding sensing and response
• Encapsulated IPTG
• Energy regeneration system
• Enzymes, ribosomes, tRNAs, cofactors
• Buffer and salts

Membrane composition

• POPC
• Cholesterol
• Maybe phospholipids to tune membrane fluidity

Encapsulated contents

• Bacterial cell-free Tx/Tl extract
• Amino acids
• Nucleotides (ATP, GTP, CTP, UTP)
• Energy regeneration substrate
• Salts and buffer
• Plasmid DNA
• IPTG

Tx/Tl system source

• E. coli (bacterial system)

Reasons:

• Simpler
• Faster
• Cheaper
• Does not require complex post-translational modifications

Communication with environment

• Input (doxycycline):

  • diffuses through membrane

• Output (IPTG):

  • released through membrane pore

• Mechanism:

  • doxycycline enters vesicle
  • activates gene expression
  • pore protein is produced
  • IPTG exits vesicle
  • bacteria detect IPTG

Experimental details

Lipids

• POPC
• Cholesterol
• Optional: DOPC

Genes (synthetic cell)

• tetR
• hla (alpha-hemolysin pore protein)

Reporter bacteria

• E. coli with:
  • gfp
  • lac promoter system

Measurement

• Measure GFP fluorescence in bacteria using:

  • flow cytometry
  • plate reader
  • fluorescence microscopy

• Controls:

  • no doxycycline
  • no pore gene
  • no IPTG
  • bacteria without reporter

• Expected result:

  • GFP only when doxycycline is present and system is functional

Homework question from Peter Nguyen

Application: Smart antimicrobial and contamination-detecting fabric

A wearable fabric embedded with freeze-dried cell-free systems that detects harmful bacteria or toxins and responds by producing a visible signal and releasing antimicrobial compounds.

How the idea works

• The fabric contains microcapsules with freeze-dried cell-free transcription/translation systems embedded into the fibers
• These systems are activated when exposed to moisture (for example, sweat, rain, or environmental humidity)
• Once activated, the system detects specific bacterial molecules using designed genetic circuits
• In response, the cell-free system produces:
  • a colorimetric signal, so basically a colour change in fabric
  • antimicrobial peptides that help reduce bacterial growth on the surface
• Because cell-free systems allow full control over components, the sensing and response modules can be easily redesigned for different pathogens or environments

Societal challenge or market need

• Rising concern about hygiene, especially in:

  • healthcare environments (hospital clothing, uniforms)
  • sportswear and everyday clothing exposed to bacteria

• Current fabrics are passive and cannot detect or respond to contamination

• This system provides:

  • real-time detection of harmful microbes
  • active response to reduce contamination
  • improved safety and hygiene without requiring complex electronics

• It could be especially useful in:

  • hospitals (infection prevention)
  • military or field work
  • travel and high-density environments

Addressing limitations of cell-free systems

• Activation with water:

  • The system is designed to activate only in the presence of moisture, making activation natural and controlled

• Stability:

  • Freeze-drying increases long-term stability
  • Encapsulation in protective polymers or hydrogels within the fabric helps preserve function over time

• One-time use:

  • The system can be designed as replaceable or layered patches within the fabric
  • Alternatively, multiple microcapsules can be distributed throughout the material so activation occurs gradually over time

• Control and reliability:

  • Because all components are specified manually, the system can be tuned for sensitivity, response time, and output intensity

Homework question from Ally Huang

Mock Genes in Space proposal

Background information (max 100 words)

Long-duration spaceflight increases radiation exposure and physiological stress, which can damage DNA and threaten astronaut health. A lightweight, freeze-dried cell-free platform is attractive in space because it is easy to transport, activated on demand, and does not require maintaining live cells. The Genes in Space toolkit specifically supports BioBits® as a “protein factory in a tube,” with miniPCR for DNA amplification and the P51 viewer for simple fluorescence readout. This makes space-based DNA damage detection both practical and scientifically valuable for astronaut monitoring, mission safety, and future deep-space habitation.

Molecular or genetic target (max 30 words)

A double-stranded DNA sequence containing a T7 promoter, GFP coding region, and defined UV/radiation-damage sites used to test whether damage reduces BioBits® expression output.

Relation of target to the challenge (max 100 words)

DNA damage can block transcription and translation, so a damaged template should produce less GFP in a cell-free reaction than an intact template. By comparing fluorescence from intact versus damaged DNA, we can estimate how strongly template integrity affects biological function in a space-compatible system. This is scientifically interesting because it connects a major space biology problem, radiation-induced molecular damage, to a direct functional readout. It also follows the logic from the recording that cell-free systems are powerful because we manually specify the parts and can rebuild and test pathways step by step.

Hypothesis / research goal (max 150 words)

My hypothesis is that DNA templates exposed to damaging conditions will produce measurably lower GFP fluorescence in the BioBits® cell-free system than matched undamaged templates. The reasoning is that radiation-related lesions can interfere with transcription by RNA polymerase and reduce the amount of functional protein produced. A cell-free system is ideal for this because it isolates the molecular effect of template damage without the added complexity of living cells, matching the recording’s emphasis on full manual control over system components. The research goal is to develop a simple, portable assay for functional DNA damage detection in space using only the Genes in Space toolkit. In the future, this approach could help evaluate DNA stability in spacecraft environments or screen protective strategies for long-duration missions.

Experimental plan (max 100 words)

I would test two DNA samples: an intact GFP template and a deliberately damaged GFP template, plus a no-DNA negative control. miniPCR would amplify the template if needed, then equal amounts of DNA would be added to separate BioBits® reactions. After hydration, reactions would incubate for protein expression, and GFP fluorescence would be measured with the P51 Molecular Fluorescence Viewer. The main data would be fluorescence intensity for each condition. Lower fluorescence in the damaged-template reaction compared with the intact-template reaction would support the hypothesis that DNA damage reduces functional gene expression in space-compatible cell-free systems.

Individual Final Project

The slide has been posted in the slide deck and I am still working on filling the form.