Week 9: Cell free systems

Week 9 — Cell-Free Systems

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. The greatest advantage the cell-free protein synthesis has over in vivo methods is that the viability of the cell does not have to be maintained in order to maintain the viability of the protein synthesis. By allowing proteins to be grown in an open environment, greater control can be exercised over the key factors that produce the protein. Materials can be added to help protein production which may otherwise be toxic to cell populations. Scientists can control ion concentrations, cofactors and energy sources. Enhancing and prohibiting materials can also be added. Growing proteins in a cell-free environment also means protein production does not have to accommodate cloning of cells.

2. Describe the main components of a cell-free expression system and explain the role of each component. I asked ChatGPT with a main prompt and then found more specific references for parts of it.¹

The main components of a cell-free expression system are:

Genetic Template. which contains the genetic information encoding the target protein. An example of a template could be a genetic circuit. The typical parts of the template would include:

• a gene promoter, which is: “…a region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene. The resulting transcription produces an RNA molecule (such as mRNA)”²
• a ribosome binding site (RBS), which is a RNA sequence found in the messager RNA to which ribosomes can bind and initiate translation ³
• coding sequence, which could be something like the sequence to code for the green fluorescent protein GFP.
• transcription terminator, which marks the end of a gene and cause transcription to stop.

Cell Extract., which contains all the machinery that will be used to support transcription and translation in the cell-free system. The machinery includes things such as:

• ribosomes, which read messenger RNA (mRNA) and translates the genetic code into a sequence of amino acids ⁴
• tRNAs, which ensure that the correct amino acid is inserted into the protein being created ⁵
• translation factors, which are proteins that “…help control when and how genes are turned on or off in a cell by binding to nearby DNA and to other DNA-associated proteins” ⁶
• metabolic enzymes which support energy procuction
• aminoacyl-tRNA synthetases, which help ensure accurate translation of genetic code ⁷

RNA polymerase. Synthesis messenger RNA (mRNA) from the DNA template during transcription. Examples include T7 RNA and SP6 RNA polymerases.

amino acids, which provide the raw materials for building proteins

Energy source. This is needed to power protein synthesis and generally comes from ATP and GTP Amino acids. These provide the raw materials to help build proteins. Nucleotides. Needed for RNA synthesis during transcription and as energy carriers during translation. ATP and GTP are important for ribosomal functions and to charge transfer RNA (tRNA). Buffers and salts. These help maintain the best conditions for supporting enzymatic activity and for stabilising ribosomes.

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.

Transcription, translation and some enzymatic reactions require great amounts of ATP, an energy-carrying molecule that resides in cells.⁸ Without them, the reaction would stop. According to Dunn, “…cells in the human body depend on the hydrolysis of 100-150 moles of ATP per day to ensure proper functioning.”⁹.

It is easier for cell-based systems to generate ATP for transcription and translation because the cells already have various metabolic networks that produce it. In cell-free systems, you have to supply that machinery.

One way of sustaining the ATP it so use a regeneration reaction such as:

ADP + Creatine phosphate –> ATP + Creatine

When transcription and translation happen, the processes get their energy from molecules such as ATP. ATP releases energy by losing a phosphate group, which turns ATP into ADP. Creatine kinase can transfer a phosphate group to help the ADP molecule go back to being ATP.

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

Whereas prokaryotic systems typically emphasise extracts from bacteria, eukaryotic systems typically come from wheat germ, insects, yeast or mammalian cells. Prokaryotic cell free platforms are typically cheaper, simpler, faster, produce higher yields. However, eukaryotic systems are better at producing membrane proteins and are better suited for therapeutic and complex proteins.¹⁰.

In a prokaryotic system I would produce sfGFP because years of work have been spent adapting and refining GFP to be produced by E. coli bacteria. I would use a prokaryotic cell free system to produce Erythropoietin (EPO), a protein used to produce red blood cells. The protein’s requirement for glycosylation isn’t well supported by eukaryotic systems.

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.

First I would determine whether the protein of interest required post-translational modifications (PTMs). Prokaryotes such as E. coli bacteria do not support PTMs that well because they lack the extra specialised cell compartments which can support modifying the protein after it is first translated. Therefore, if the protein did not need them I would focus on the simpler eukaryotic system. Otherwise I would focus on using a prokaryotic-based cell-free system.

Membrane proteins can be difficult to synthesise in cell-free systems because there is no membrane that could influence how they well they fold or their solubility. Therefore, the systems need to support materials that can mimic a membrane. Zemella lists nanodiscs, liposomes and certain detergents that have been developed to mimic the missing membrane. ¹⁰

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

Three causes of low yield for target proteins in a cell-free system include:

• inefficient transcription or translation.
• protein misfolding
• energy depletion

Inefficient transcription or translation may be caused by different causes, which include:

• weak promoter that doesn’t activate translation of downstream genes as well as it could
• the ribosome has difficulty binding to the RBS

Choosing a stronger promoter can mean the promoter switches transcription ‘on’ more often. Inserting filler base pairs round the RBS sequence can make it easier for the RBS to stand out and be grasped properly by the ribosome (like I put into my final project Twist sequence).

Misfolded proteins could be caused by various factors such as the proteins containing hydrophobic regions. In this case, the use of detergents, nanodiscs or liposomes could help. As Puthenveetil notes: “Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques.” ¹¹

The system could run out of supplies materials such as ATP, GTP, amino acids and magnesium that help provide the cell-free system with energy. It could also develop byproducts which could stall translation prematurely. One solution is to try to reduce these byproducts and another is to increase inputs such as ATP, amino acids and magnesium.

Homework question from Kate Adamala

1. Pick a function and describe it.

1a. What would your synthetic cell do? What is the input and what is the output?

Consider a synthetic cell that is capable of fluorescing when it detects the presence of lactose.¹². The system’s input would be a lactose molecule that resides outside the synthetic cell and the output would be the Green Fluorescent Protein (GFP).

1b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation? Yes, it could be done in a cell-free transcription/translation environment. It may even be preferable to using a solution with encapsulation, unless a project called for the biosensor cell to support other functions besides just sensing lactose. Then there may be a need for a synthetic cell to support different functions in different compartments.

1c. Could this function be realized by genetically modified natural cell? Yes.

1d. Describe the desired outcome of your synthetic cell operation. When a lactose molecule passes through the membrane pores of the synthetic cell, it can bind to a regulatory protein such as Lac and signal that transcription of the GFP protein should begin.

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

The cell would comprise three modules:

• a sensing module, which could be a plasmid genetic circuit
• a gene expression module that uses PURE (Protein synthesis using Recombinant Elements) system
• an output module that transcribes and translates the Green Flourescent Protein inside a vesicle

2a. What would be the membrane made of? I would use phospholipids because they are closest chemically to real biological membranes.

2b. What would you encapsulate inside? Enzymes, small molecules. I would include the following systems:

• the transcription-translation system made from PURE components
• an energy system to sustain transcription and translation
• small molecules used for protein synthesis
• enzymes

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 Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason?need mammalian) Bacteria is OK to use because lactose sensing is a prokaryotic regulatory feature. For example, the lactose operon exists in E. coli and contains genes involved with metabolising lactose ¹³. You could use a mammalian system but it would add more complexity without adding much benefit.

2d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?) In order for the circuit to work, the GFP protein would need to be able to pass out of the membrane of the synthetic cell. One way to help that happen is to ensure the synthetic cell has generic pores that could transport it.

3. Experimental details. 3a. List all lipids and genes. A common set of lipids includes:

• Phosphatidylcholine (PC)
• Phosphatidylethanolamine (PE)
• Phosphatidylglycerol (PG)

The lactose repressor gene would be the prominent gene in this system.

3b. How will you measure the function of your system? I would measure the amount of green fluorescent protein (GFP) produced by using flow cytometry.

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.

1. Write a one-sentence summary pitch sentence describing your concept. My idea would be to develop a bite-proof body suit that would fluoresce with colour if it detected the presence of saliva from Ixodes ricinus, the tick that carries the Lyme disease bacteria in Europe.¹⁴.

2. How will the idea work, in more detail? Write 3-4 sentences or more. Community health specialists who are assessing the risk of getting bitten by Lyme disease ticks on walking trails and fields could use the body suit to measure realistic exposure without getting bitten. A cell-free system would be integrated into the outer layer of clothing designed to prevent tick bites from penetrating the skin. The cell-free system would detect salivary protein IrAV422, which would be activated when the dried cell-free system encountered saliva from the bug. The system would then manufacture a fluorescent protein in the area of the bite attempt. The system would include peptides that could bind IrAV422 and trigger the production of the fluorescent protein.

3. What societal challenge or market need will this address? As weather patterns change, Lyme ticks are able to live in a wider range for longer during the year. Lyme disease isn’t always obvious to detect and it would be a good way to show people where ticks were detected.

4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? I do wonder how effective such a system would be in sensing from such a small amount of saliva from the bug. The clothing may wear out and I would imagine putting it through a washing machine could severely degrade it. Perhaps it would be designed as a cheap, biodegradable fabric that is meant to be used only a few times until it needed to be washed.

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)

How do tardigrades respond physiologically to extreme environment stressors such as radiation, extreme temperature and a lack of oxygen? This topic could be important for understanding genes that could be useful in making microorganisms that could accomplish important tasks in inhospitable environments. I would be designing cell-free systems which could help monitor expression of proteins that helped it survive. This could be important for transporting biological materials when there is limited payload space available for systems to keep living organisms alive.

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 focus on genes that code for Dsup, a protein that helps tardigrades survive severe radiation.¹⁵

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) I would focus on the damage suppressor protein Dsup, which helps tardigrades survive extreme conditions.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) My hypothesis would be that if you integrated a cell-free system that detected Dup inside a microfluidics channel that housed the tardigrade, it would be expressed more in extreme 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) I would create clones of tardigrades and then establish various experiments that used a control and an extreme condition. The control would correspond to conditions it enjoyed on Earth that would also be favourable to humans. For the extreme condition (e.g. extreme heat, cold, vacuum, radiation), I would use the cell-free system to help detect the presence of the Dsup protein (e.g. fluoresence, electrical signal). I would then measure each indicator between control and extreme condition. I would expect the extreme condition to reflect higher Dsup production.

Homework Part B: Individual Final Projec

Done