Week 8 HW: Cell-Free Systems

General HW 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 allow the expression of systems that might otherwise be toxic to or impose a high level of burden upon a living cell. The lack of native cellular pathways (which often respond to circuit presence in unexpected ways and can sometimes compromise the functioning of a circuit) also means that the researcher need not fear interference or complications due to the behavior of the host. Cell free methods are more beneficial than cell production for working with sensing/detection constructs such as riboswitches, since (in the absence of a cell membrane) the construct will function better with direct access to compounds of interest in the environment. Cell free methods are also advantageous when studying systems that would otherwise kill or significantly reduce the functioning of the host cell – e.g., toxins or high-burden circuits.

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

Cell free systems contain:

• Ribosomes and tRNAs: To provide the mechanisms necessary for translation
• ATP: To power translation
• mRNA: To be translated –> Note that you can either use existing mRNA or provide DNA and RNA polymerase to transcribe it
• Any enzymes necessary for the pathway being expressed

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.

Energy provision is critical in cell free systems because it powers transcription and translation. Cell-free systems typically provide a continuous ATP supply through specific kinase and phosphatase-based enzymatic pathways.

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

Prokaryotic cell-free systems use prokaryotic cell lysates and are faster and cheaper than eukaryotic cell-free systems. Eukaryotic cell-free systems use eukaryotic cellular machinery, allowing the expression of eukaryotic proteisn that require processing steps involving eukaryote-specific machinery, such as the endoplasmic reticulum and golgi apparatus. Some examples of proteins optimal to express in each system are: In prokaryotic lysates – specific bacteriocins, anti-microbial proteins produced by bacteria to kill competitor bacteria; In eukaryotic lysates – specific antibodies, which usually require folding/processing in the eukaryotic endomembrane system.

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.

To assess and optimize a membrane protein’s expression, I would create a cell free system that includes relevant cell membrane components. Membrane proteins’ funcitoning is highly dependent on protein structure and on the hydrophobicity and hydrophilicity of the proteins’ domains, so I would optimize the environment (e.g., ion concentrations) for effective folding.

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.

The low yield could be because:

1. The circuit expressing the target protein was flawed

• switch to a stronger promoter or from an inducible to a constitutive promoter
• validate circuit sequence / identify issues via sequencing

2. The system did not include enough of one or more required cellular components

• test the system in an excess of amino acids, tRNAs, ribosomes, ATP, etc

3. The protein’s complete synthesis is dependent on the presence of other proteins that aren’t present in the current cell free system

• understand the complete pathway for the protein’s syntheis and add relevant cellular components as necessary

Questions from Kate Amadala

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?

My synthetic minimal cell would express the Mcy toxin-production gene cluster in harmful algal bloom-causing cyanobacteria. Given my minimal cell would presumably have a substantially reduced genome, its degree of functioning or non-functioning would clarify the minimimum genetic conditions required for toxin production.

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

While this could potentially be realized by TxTl, encapsulation is more reflective of a real cellular environment (/ provides the minimum conditions of a cellular environment) and would provide potentially more-relevant/applicable information about the gene cluster’s funcitoning.

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

Yes, this function could be realized through gene-knockout studies. However, cyanobacteria are difficult to genetically engineer, and gene knockout in real cyanobacterial cells might not be feasible to perform at a large enough scale for high throughput screening.

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

My synthetic cell should provide a model that I can use for genetic screening to assess the effects of various genetic modifications (and other changes) on Mcy performance and expression – to ultimately identify protein targets for anti-microcystin and anti-HAB strategies.

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

a. What would be the membrane made of?

The membrane would replicate that of a gram negative bacterium: with an outer phospholipid membrane, a peptidoglycan layer, and an inner membrane.

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

A minimal or reduced genome, ribosomes, tRNAs, ATP.

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)

Bacterial. Potentially, Microcystis aeruginosa.

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

My synthetic cell will likely need to express membrane channels necessary for basic ion homeostasis.

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.)

• Minimal M aeruginosa genome
• Mcy genes A-J

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

Lyse cells and use a toxin detection assay to measure microcystin production. (And potentially use other chemistry techniques to assess its chemical structure.)

Questions from Peter Nguyen

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.

I propose a face mask that performs real-time virus detection via a cell-free system embedded in the fabric.

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

The face mask will contain a cell free system consisting of several detection systems for multiple pathogens. Potentially, the cell lysate could contain multiple riboswitches that detect pathogen genetic material and express a reporter of a color that corresponds with the pathogen being detected. (Note that the cell-free system might require a built-in “disruption” step to get viral genetic material out of the capsid, which may not be feasible )

• What societal challenge or market need will this address?

The product will make real-time at-home multi-pathogen testing more accessible and encourage users to seek medical treatment when necessary and avoid spreading infectious disease.

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

This product would likely have to be one-time use only – or could potentially be used until a positive test. To compensate for the sustainability downsides of such a setup, the cell free component could be replaceable – embedded in a reusable cloth mask.

Questions from Ally Huang

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/ .

1. 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)

How does the performance of well-characterized genetic circuits (e.g., the repressilator and toggle switch) differ under space conditions / aboard a space station as compared to a traditional Earth-based laboratory setting? Some space conditions for testing include: microgravity, radiation exposure, low-oxygen, and vacuum exposure. Understanding how circuits behave in these contexts–independently of a living cell/chassis, which contains complex machinery that would likely fail and compromise circuit performance under many of these conditions–would help us optimize engineering and circuit design strategies for space applications (developing a set of SynBio “design principles” for space).

2. 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)

I would study existing well-characterized genetic circuits such as the Repressilator and Toggle Switch. I might also look at very simple systems expressing only a reporter, and potentially with varying control systems (constitutive expression vs. inducible expression, etc).

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

The above circuits would allow me to easily benchmark their performance under space conditions via comparison with their performance under typical laboratory conditions. I would use RNA sequencing and analyze reporter expression via spectrophotometry and other fluorescence measurements in order to identify differences in performance and characterize performance under different space-like conditions.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

My research goal is to characterize circuit performance (in cell-free systems) under a variety of space-like conditions, varying the circuit design and space conditions of interest. My results will inform future cell-free system applications in space and will also generate foundational knowledge about the effect of space on circuit behavior–independently of a bacterial chassis.

5. 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 will recreate the repressilator and toggle switch experiments, adapted to use cell free expression rather than bacterial cells, under several space-like conditions: 1) a microgravity simulator, 2) under some form of radiation, 3) under low oxygen conditions, and 4) a normal Earth laboratory setup (the control). I will collect fluorescence measurements at multiple timepoints to assess whether circuit behavior differs from the control under the different conditions. I will also perform RNA sequencing to assess the timing of gene expression of the circuits within the cell free systems and determine whether it varies across conditions.