Week 9: Cell-Free Systems

Part 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.

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.

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.

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.

Part 2: Questions from Kate

Design example of useful synthetic minimal cell.

1. Pick a function.
2. Design all components that would need to be part of your synthetic cell.
3. Experimental details

Example solution given below, based on: Lentini, R. et al., 2014. Nat comm, 5, p.4012.

1. Pick a function What would your synthetic cell do? What is the input and what is the output. Expand the sensing capacity of bacteria. Input: theophylline (inert to bacteria). Output of the SMC: IPTG. Output of the whole system: GFP produced in bacteria.

Theophyline Aptamer reference: Martini, L. & Mansy, S.S., 2011. Cell-like systems with riboswitch controlled gene expression. Chemical Communications, 47(38), p.10734.

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

No. If the IPTG was not encapsulated, it would go into the bacteria without the need of theophylline-induced membrane channel synthesis, thus the synthetic cell actuator would not exist.

Could this function be realized by genetically modified natural cell? Yes, in this particular case: theophylline aptamer could be incorporated into a transformed gene. This lacks generality though – it is easier to make SMC than modify bacteria, so in this system a single bacteria reporter can be used to detect various small molecules.

Describe the desired outcome of your synthetic cell operation. In the presence of SMC, bacteria sense theophylline.

2. Design all components that would need to be part of your synthetic cell. 2A What would be the membrane made of? Phospholipids + cholesterol.

2B What would you encapsulate inside? Enzymes, small molecules. Cell free Tx/Tl system, IPTG, gene for membrane transporter under the control of theophylline aptamer.

2C 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, because of the theophylline riboswitch used as SMC input.

2D How will your synthetic cell communicate with the environment? (hints: are substrates permeable? or do you need to express the membrane channel?)

The membrane is permeable to the input molecule (theophylline), the output is IPTG that will cross the membrane via the membrane pore created after theophyline-initiated gene expression.

3. Experimental details

3A List all lipids and genes (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick actual gene)

Lipids: POPC, cholesterol

Enzymes: bacterial cell free tx/tl

Genes: a-hemolysin (aHL) to encapsulate in SMC,

Biological cells: E.coli transformed with GFP under T7 promoter and a lac operator

3B How will you measure the function of your system?

Measure GFP output of the cells, via flow cytometry. Alternatively, use enzymatic reporter, like luciferase, and measure bulk output of the enzyme.

Artificial cells translate chemical signals for E. coli.

(a) In the absence of artificial cells (circles), E. coli (oblong) cannot sense theophylline.

(b) Artificial cells can be engineered to detect theophylline and in response release IPTG, a chemical signal that induces a response in E. coli.

Part 3: Questions 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. How will the idea work, in more detail? Write 3-4 sentences or more. What societal challenge or market need will this address? How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Part 4: Questions 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) 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) 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) 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)