Week 9 HW: Cell-Free-Systems

Homework Part A: General and Lecturer-Specific Questions

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.

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

  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.

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

  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.

  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.

Homework questions from Kate Adamala

  1. Pick a function and describe it.

  • What would your synthetic cell do? What is the input and what is the output?

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

  • Could this function be realized by genetically modified natural cell?

  • Describe the desired outcome of your synthetic cell operation.

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

  • What would the membrane be made of?

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

  • 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)

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

  1. Experimental details

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

  • How will you measure the function of your system?

Example solution

Based on Lentini, R. et al., 2014. Nat comm, 5, p.4012.

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 were 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 a genetically modified natural cell?

Yes, in this particular case: the 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.

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

What would be the membrane made of?

Phospholipids + cholesterol.

What would you encapsulate inside? Enzymes, small molecules.

cell-free Tx/Tl system, IPTG, gene for membrane transporter under the control of theophylline aptamer.

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.

How will your synthetic cell communicate with the environment? (hint: 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.

Experimental details

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

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.

Homework 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.

Water-producing rescue robots: earthquake rescue robots designed with a chemotactic sensing ability to detect live human presence, to inform, and to deliver water harvested from the atmosphere to survivors.

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

There would be two main cell-free systems in the robot:

  1. Breath detection for the living and separation of the deceased by the absence and presence of certain volatile compounds, which would allow making decisions by using logic gates with specific sensing circuits (Example: AND-gate; CO₂ + acetone; OR-gate; isoprene or CO₂ live human detected).

    As the cell-free system is freeze-dried, it needs to be hydrated and activated. This can be achieved through a hydrogel as described below. Additionally, the robot’s movement needs to be directional and responsive to a gradient of the signals in order to locate survivors. Similar to a bacterial chemotaxis system, which resets and generates a new response in the gradient of chemicals, a robot’s sensing circuits can be designed to reset, aiding the movement in a gradient.

  2. Water harvesting hydrogel: The robot has a hydrogel that harvests moisture from the atmosphere continuously. An enzyme that can cleave the hydrogel matrix, such as a protease or cellulase, could be activated by CO₂ at a threshold (or other breath volatiles). Water is released from the hydrogel. Then, water is collected in a tube.

  • What societal challenge or market need will this address?

An earthquake is not known in advance. Building collapses, trapping people in rubble, are unavoidable. The water-producing rescue robot could extend the time for survivors as help is on the way.

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

Because hydrogel traps water, it is available for hydration. A microfluidic delivery device can connect the two, hydrating cell-free freeze-dried components on demand. The robot’s directional movement would be dependent on the shelf life of the activated enzymes. A built-in camera, GPS, etc., should be helpful for informing the location of the robot.

Homework question 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 link.

  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)

  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)

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

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

  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)

Homework Part B: Individual Final Project

  1. Put your chosen final project slide in the appropriate slide deck.
  2. Submit Final Project selection form.
  3. Begin planning how you will write your final project documentation based on these guidelines.
  4. Prepare your first DNA order.

Below is the final project slide screenshot.

This project computationally addresses aim 1.

Fig Fig