Week 9 HW

Homework Part A: General and Lecturer-Specific Questions

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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 Question from Kate Adamala

Prompt

Design an example of a useful synthetic minimal cell as follows.

Function

  1. Pick a function and describe it.
  2. What would your synthetic cell do? What is the input and what is the output?
  3. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
  4. Could this function be realized by a genetically modified natural cell?
  5. Describe the desired outcome of your synthetic cell operation.

Synthetic Cell Components

  1. Design all components that would need to be part of your synthetic cell.
  2. What would the membrane be made of?
  3. What would you encapsulate inside? Enzymes, small molecules.
  4. Which organism will your Tx/Tl system 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 promoters, like Tet-ON, you need a mammalian system.

  1. How will your synthetic cell communicate with the environment?

Hint: Are substrates permeable, or do you need to express the membrane channel?

Experimental Details

  1. List all lipids and genes.

Bonus: Find the specific genes. For example, instead of just saying “small molecule membrane channel,” pick the actual gene.

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

Example Solution

Based on: Lentini, R. et al., 2014. Nature Communications, 5, p. 4012.

Pick a function and describe it.

Expand the sensing capacity of bacteria.

  • Input: theophylline, which is inert to bacteria.
  • Output of the synthetic minimal cell: IPTG.
  • Output of the whole system: GFP produced in bacteria.

Theophylline 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 enter the bacteria without the need for theophylline-induced membrane channel synthesis. In that case, 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. However, this lacks generality. It is easier to make synthetic minimal cells than to modify bacteria, so in this system a single bacterial reporter can be used to detect various small molecules.

Describe the desired outcome of your synthetic cell operation.

In the presence of synthetic minimal cells, bacteria can sense theophylline.

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

Membrane

  • Phospholipids
  • Cholesterol

Encapsulated components

  • Cell-free Tx/Tl system
  • IPTG
  • Gene for membrane transporter under the control of the theophylline aptamer

Which organism will your Tx/Tl system come from?

Bacterial, because the system uses the theophylline riboswitch as the synthetic minimal cell input.

How will your synthetic cell communicate with the environment?

The membrane is permeable to the input molecule, theophylline. The output is IPTG, which crosses the membrane through the membrane pore created after theophylline-initiated gene expression.

Experimental details

Lipids

  • POPC
  • Cholesterol

Enzymes

  • Bacterial cell-free Tx/Tl

Genes

  • α-hemolysin, or aHL, to encapsulate in the synthetic minimal cell

Biological cells

  • E. coli transformed with GFP under a T7 promoter and a lac operator

How will you measure the function of your system?

Measure GFP output of the cells using flow cytometry. Alternatively, use an enzymatic reporter, such as luciferase, and measure the bulk output of the enzyme.

Figure caption

Artificial cells translate chemical signals for E. coli.

  • In the absence of artificial cells, E. coli cannot sense theophylline.
  • Artificial cells can be engineered to detect theophylline and release IPTG in response.
  • IPTG then induces a response in E. coli.

Homework Question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into many kinds of materials as biological sensors or as inducible enzymes that modify the material itself or the surrounding environment.

Choose one application field:

  • Architecture
  • Textiles/Fashion
  • Robotics

Then propose an application using cell-free systems that are functionally integrated into the material.

Answer each of these key questions for your proposal pitch:

  1. Write a one-sentence summary pitch describing your concept.
  2. How will the idea work, in more detail? Write 3–4 sentences or more.
  3. What societal challenge or market need will this address?
  4. How do you envision addressing the limitations of cell-free reactions, such as activation with water, stability, or one-time use?

Homework Question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in the lecture, 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 high school students, this assignment asks you to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space.

For this assignment, your proposal must incorporate the BioBits® cell-free protein expression system. You may also use other tools in the Genes in Space toolkit, including:

  • miniPCR® thermal cycler
  • P51 Molecular Fluorescence Viewer

For more inspiration, see: https://www.genesinspace.org/

Proposal Questions

  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 or samples you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, and any relevant workflow details.
    Maximum: 100 words.