Week 9 HW: Cell-Free Systems

Homework Part A

Assignment

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.

It lacks the stress response, proteostasis and detoxification of an in vivo environment, allowing for much higher expression of protein including toxic varieties or those incorporating xenobiotic compounds, as in bio-orthogonal experiments. Reaction conditions are standard and can be externally controlled/varied (eg temperature) more precisely than in in vivo systems. The progress of reaction is more easily followed using imaging techniques, allowing for rapid optimization and sequential syntheses. Two cases where cell-free expression is better than cell production are the expression of protein toxic to bacterial/fungal chassis (eg antibacterial/antifungal compounds) as well as proteins intended for use in human medicine (free of endotoxins).

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

  • Cell Extract (specifically, transcription and translation machinery): Allow for the transcription and translation of DNA input
  • ATP/GTP as well as phosphorylative regeneration system: Energy carrier drives the expression of protein
  • Nucleotide bases (ie NTPs): For transcription of DNA to form mRNA
  • Nucleic acid template: Encode protein/construct to be expressed
  • Buffer solution: Provide necessary salts, cofactors and buffers in an isosmotic system for optimal protein expression

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.

Cell-Free systems do not natively provide for substrate-level or oxidative phosphorylation required for ATP/GTP regeneration. ATP/GTP in turn is required for the endergonic processes involved in expression (eg GTP-driven ribosomal translation of primary transcript; ATP-driven tRNA aminoacetylation). I would include excess creatine phosphate to function as a Pi donor and creatine kinase to catalyze the phosphorylation of ATP/GTP in the cell-free system buffer solution.

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

Prokaryotic cell-free expression systems have higher yields of final product, are faster and less complex than eukaryotic cell-free expression systems, though the latter enable the production of larger, more complex proteins complexed with cofactors and coenzymes.

Consider the production of GFP in a prokaryotic system: simultaneous transcription and translation means multiple GFP polypeptides may be synthesized in parallel, while the autocatalytic folding of the protein removes the need for post-translational modification or energetically costly chaperonins.

Now consider, alternatively, the production of selectin in a eukaryotic system: given the glycoprotein nature of selectin, after translation yields the primary structure of selectin, it undergoes post-translational modification to have a glycosidic component linked chemically.

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.

After identifying the key components of the reaction mix and how they might impact expression (eg the relative concentrations of ions in the buffer, buffer pH, relative concentrations of GTP/ATP and components of the phosphorylative regeneration system), I would vary these conditions across a 96-well plate in triplicate and incubate the plate under constant conditions. At a fixed endpoint, I would quench the reaction and use spectrophotometry to determine the respective expression efficiency in each well, and thus the reaction conditions which produced the highest protein expression levels.

One challenge could be the imprecision of reaction mix formulation. I would use lab automation to reduce variation in reaction mix preparation and minimize error.

Another challenge could be a lack of standard lysate. I would prepare a large enough stock of lysate prior to experimentation.

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.

  1. Poor Quality of DNA template: Unoptimized codons and endonuclease contamination could reduce the efficiency of transcription and translation. Troubleshooting: Using the same preparation and reaction mix, substitute a known optimized plasmid for the DNA template provided.

  2. Insufficient ATP/GTP regeneration: ATP/GTP are rate-limiting due to the slow rate/insufficient regeneration Troubleshooting: Repeat the experiment with an excess of creatine phosphatase and kinase

  3. Insufficient cofactors: cofactors such as ${Mg}{2+}$ are rate-limiting due to a low concentration in reaction buffer which reduces the rate of formation of holoenzymes required for transcription and translation Troubleshooting: Repeat the experiment with an excess of cofactors (eg. ${Mg}{2+}$) added to the reaction mix.

Homework question from Peter

Freeze-dried cell free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field - Architecture, Textiles/Fashion, or Robotics, and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

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

Imagine a smart layer you could add to windows to filter out UV rays in sunlight but which self-healed and wasn’t subject to photo-degradation.

* How will the idea work, in more detail? Write 3-4 sentences or more.

A cell-free system incorporated in a hydrogel will express photoprotective protein (eg. lycopene) under the regulation of a microfluidic logic chip. The logic chip could be hard-wired for a particular region’s climate, or incorporate a microprocessor to allow real-time updates from a photosensor. The lycopene itself will be degraded in the sunlight, but offer bespoke UV protection which compared to traditional UV films will be regenerable, allow vitamin D-promoting wavelengths and the natural warmth of sunlight through, and allow fine customization of which wavelengths of sunlight get filtered out.

* What societal challenge or market need will this address?

This would help minimize energy used for cooling as the world becomes more urbanized and meet the need for window conditioning in construction.

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

Within the hydrogel film, the raw materials (eg rainwater) will be collected from the environment itself and stored in reservoirs which the microfluidic logic then transports to a well-protected reaction core housing the cell-free system proper.

Homework question from Ally

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/

Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words)

The risk of illness in long-duration human spaceflight is unconscionable; not being able to diagnose and consequently treat mystery illnesses only more so. I aim to provide a low-cost battery of diagnostics to rapidly identify pathogens, with strong translational potential for rural healthcare delivery on Earth.

Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words)

Metabolomics.

Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

Clearly state your hypothesis or research goal and explain the reasoning behind it (Maximum 150 words) All pathogens carry metabolomic signatures which specifically identify strains, types and serotypes. My approach allows for a broad-range of pathogens to be identified without heavy, high-maintenance laboratory equipment.

Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

I’ll use 3 samples of E. coli, T4 Phage and C. elegans to simulate bacterial, viral and nematodic pathogens in a BSL-2 environment. DI will be a -ve control. My experiment will involve the multi-repeat testing of a cell-free metabolomic screening system across serial dilutions of pathogen titers to establish diagnostic specificity and sensitivity.

Homework Part B

https://docs.google.com/document/d/1lCuoHWFwwyRXwhZk5J6fBLDnvC8MFbETRV1QUFZBLPo/edit?tab=t.0