Week 9: Cell-Free Systems

Week 9: Cell-Free Systems

Homework — DUE BY START OF Apr 7 LECTURE

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.

Cell-free systems help us understand biology ‘from scratch’ to bioengineer from smaller units. There’s wider flexibility for scaffolding biology from the ground-up and controlling the environments in a complete model. Existing living cells as we know it are already incredibly complex and hence less controlled in experimental settings. Synthetic cell engineering allows flexibility in size of the cell, proteins, and even expanding largely on the chemistry of the cell. So the two scenarios could be if you want to control the size of the cell and want uniform control it might be ideal to use cell-free system. The other scenario might be to engineer a specific chemical environment or want chemical diversity in the experiment that is not naturally common/ compatible with cells. Compared to in-vivo expression where you have to create plasmids, cell-free protein expressions are faster and cheaper to construct and can also help you through quick iterations with linear fragments and without plasmids.

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

The anatomy of the synthetic cell has multiple parts:

1. phospholipids and cholesterol to create strong lipid membranes
2. cytoplasm contains small molecules
3. cell extract such as ribosomes and enzymes
4. tRNAs
5. plasmids and membrane channels for communciation

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.

Within normal cells, energy is continuously regenerated through metabolism, but cell-free systems are normally carried within microfluidics or vesicle and isn’t able to have the same glucose-ATP interactions a normal cell does. To achieve continous protein synthesis we must also introduce additional energy substrates and enzymatic regeneration systems. Common practices include introducing either phosphoenolpyruvate (PEP), creatine phosphate (CP), or acetyl phosphate (AcP) for rapid ATP regeneration via kinases present in the extract.

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

Transcription happens in nucleus for eukaryotes but in cytoplasm for prokaryotes.

Within prokaryotic cell-free systems, transcription and translation happen at the same time, they are much faster and productive. In contrast eukaryotic systems separate (exons and introns) and will require specific machinery enzymes that will take out the introns. They retain ribosomes, chaperones, and modification enzymes so that there is correct folding and processing of complex proteins.

GFP can be produced in a prokaryotic system and commonly produced in E Coli lysate, it is small and does not require glycosylation.

Complex human proteins are more appropriately made in eukaryotic cell free systems. Membrane proteins such as GPCRs are usually expressed through wheat germ extract. Because they are hydrophobic and are 7 transmembranes, it is difficult to fold while inserting into a membrane and require a lipid bilayer.

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.

Membrane proteins like GPCRs are difficult because they require eukaryotic chaperones to correctly fold. Bacterial systems like E Coli lysate do not have the machinery to handle hydrophobic transmembrane domains without aggregation.

To avoid challenges like aggregation, liposomes or nanodiscs might be added to the raction mixtures so that we can help with co-translational membrane insertion.

Another problem with cell free systems is that it’s hard to distinguish protein from everything else in the mixture, so using His-tags are very useful ways to pull out specific protein using histadine and wash out other components.

Fusion protein
  |
His-tag (histadine)
  |
Ligand
  |
Bead 
------
Magnetic block 

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.

First it may be that ATP depletes quicker than it regenerates. In this case we will need to switch from PEP to other systems like creatine kinase for slower reactions.

Secondly, there might be aggregation due to hydrophobicity of the protein, these chains that are poking out that normally can embed into a nearby membrane will end up clumping together with other hydrophobic sections, leading to misfolding. Again Nanodiscs might help to provide hydrophobic environment so that proteins can bind to these discs as opposed to binding into each other.

mRNA is also unstable. Cell free lysates will degrade the mRNA template quickly and so maybe there might not be enough translation occurring. RNase inhibitors are typically used to stabilize.

Homework question from Kate Adamala

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

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

   I am interested in a synthetic minimal cell that may act as an artificial dopaminergic synapse sensor, so the input will potentially be extracellular dopamine released by differentiated PC12 cells, and to make this signal visible, we will also need a GFP or RFP florescent signal to identify proportional to dopamine concentration and report a reward signal. Typically, this must be done with encapsulation, particularly due to how membrane proteins play a huge role in allowing dopamine receptors through GPCR and that DRD1 must be expressed with the presence of lipid environment. So one way of working with this is a eukaryotic cell free system with the introduction of liposomes or nanodiscs directly into the cell-free reaction. Yes, for part of the final project I am working on this exact function via overexpressing DRD1 gene with GFP construct in PC12 cells. But natural cells have hundreds of competing pathways activated by dopamine and it’s easier to control density/ concentrations in a synthhetic cell. The desired outcome is that the synthetic minimal cell can successfully operate as a dopamine-responsive optical reporter and report with florescence that maps dopamine release events.

1. What would be the membrane made of?
2. What would you encapsulate inside? Enzymes, small molecules.
3. 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)
4. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

The membrane will likely be a cholesterol liposome bilayer. Since GPCRs insertions require cholesterol-rich membranes to adopt correct conformation for DRD1 to express, cholestrol can help increase membrane rigidity and support that insertion. For the transcription and translation machinery, we might want to use HEK293 cell-free lysate system with T7 RNA polymerase for in-vitro transcription. The DNA constructs will include a DRD1 insertion plasmid and GFP construct embedded or separated with cAMP response signalling sequence. We need mammalian set-up because GPCR is a membrane protein. Using HEK293 lysate cell free system retains glycosylation that DRD1 needs to be expressed. It will communicate with the environment via cAMP signalling, as dopamine binds extracellular DRD1 and will trigger intracellular cAMP signalling without requiring membrane permeation.

3. 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.)
   2. How will you measure the function of your system?

POPC (Palmitoyloleoylphosphatidylcholine), DOPG (Dioleoylphosphatidylglycerol ), and Cholesterol for lipids to create liposome bilayer. Genes: DRD1, protein kinase, cAMP Response Element Binding Protein 1 (CREB1), EGFP for florescence, T7 RNA Polymerase (ecoli phage). Using a plate reader we will have a range of different concentrations and read florescence. The Plate reader fluorescence assay will allow us to run 96-well plate and add extracellular dopamine at different concentrations 0, 1nM, 10nM, 100nM, 1µM, 10µM alongside lipid bilayer POPC:DOPG:Cholesterol = 60:10:30 mol%. Then we can measure GFP florescence every 30 min.

Homework question 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.
• 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)?

I would like to design a biosensor for robotics ‘in the wild’, where only under specific weather conditions it will use freeze dried cell free system for repair or environment-adaptive changes. For example, performing localised self-repair protein expression on the skin. To think about robotics as regenerative rather than designed and completed ‘at a factory’. Just like a tub of instant coffee that can be used ‘on tap’- the fact that it can be activated with water is extremely stable!

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 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)

There’s actually a lot of research right now out of UC San Diego via Alysson Muotri on brain organoids in space. There’s growing research and need to maybe thinking about hybrid brains or neural repair in space that might be useful in rescuing living substrates mid-journey? Neural surgery performed in brain organoids or to support fusion in space also means that we will need to do bioprinting or scaffolding using cell-free systems via lab robot.

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 am interested in DRD1 dopamine receptor D1, Forkhead Box A2 and Nurr1 transcription factors that regulate and amplify dopamine reception in cells. We could do more chemical signalling and reinforcement if we are able to make these cells more receptive to signals.

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

DRD1 is the primary receptor mediating dopamine’s effects on motivation, working memory, and motor coordination. By expressing DRD1 in a BioBits cell-free system with fluorescent reporter, we can either detect dopamine concentration in biofluid samples or test receptiveness of cells in space experiments.

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

The hypothesis will be centered around whether dopaminergic functions will be retained after freeze-drying and rehydration under simulated microgravity conditions.

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)

It could be a comparative experiment against freeze-dried samples with rehydration against living samples to test if freeze-drying retains dopeminergic functions. Since PC12 cell-based dopamine research on Earth requires living cell cultures, CO2 incubators, freeze-drying will streamline and potentially use as neurochemical monitoring toolkit.

Homework Part B: Individual Final Project

Check final project page!