Week 9 HW: Cell Free Systems

HTGAA Week 9 Cell Free Systems

Part A General and Lecture 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.

Time : CFPS can be executed in very little time, a couple of hours however in vivo methods will take a few days to a few weeks. Time is a key part of efficient research as less time is wasted waiting to synthesize proteins to experiment on.
System : CFPS is an open access system as it has no membrane and gives direct access to the molecule which one wants to work on, it offers more flexibility. It also allows for better control over the synthesis as one can specifically choose which components to input in a protein and better predict protein folding, this simplifies monitoring as well. Versus an in vivo method which has a membrane and therefore a closed system, this means the host and its other components will also react to any modification making it harder to control and direct, the cell’s functioning can often get in the way of the synthesis or create unpredicted issues.
Tolerance: CFPS can tolerate high rates of toxic and difficult proteins as it is non living, however, in vivo technologies are more sensitive to toxins as it is likely to harm the host. CFPS also works using non-natural components offering a wider spectrum of possibilities.
Complexity : CFPS is a simple PCR based procedure while in vivo requires more complex cloning and transformation steps.

Reference List Silverman, A. D., et al. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics. Gregorio, N. E., et al. (2019). Cell-free microbial synthesis of proteins. Frontiers in Bioengineering and Biotechnology.

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

There are four core components to a CFPS system which are the catalytic engine, the instruction template, the energy source and the biochemical blocks.

At first a “soup” is created by breaking a cell pen and removing debris, this gives us the heart of the system which provides us with the molecules required for transcription and translation like ribosomes or tRNAs. The DNA instruction template is the blueprint for the CFPS and it is very flexible as it can function with a plasmid but it can also work using PCR linear PCR products which allows to reduce cloning time in the preparatory stages. Compared to most synthesis technologies requiring a lot of energy to keep the cell alive and the ribosomes active, CFPS can be fueled by ATPs and GTPs which are high energy sources. Biochemical building blocks and buffers stabilize the CFPS reaction by adding to the solution amino acids to build the chain, the RNA polymerase and often magnesium or potassium salts which can help stabilize the protein folding.

Reference List Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics. Hodgman, C. E., & Jewett, M. C. (2012). Cell-free protein synthesis: The state of the art. Biotechnology and Bioengineering. Caschera, F., & Noireaux, V. (2014). Synthesis of 2.3 mg/ml of GFP with an all-E. coli cell-free transcription-translation system. Biological Engineering. Shimizu, Y., et al. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology. Tinafar, A., et al. (2019). A Manual of Cell-Free Protein Synthesis Systems. Frontiers in Bioengineering and Biotechnology.

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.

Protein synthesis is a very energy taxing process. An issue in CFPS is the ATP “leaks” caused by enzymes breaking down enzymes to ADP (without the ribosome using it) and inorganic phosphate, the regeneration of energy allows to preserve fuel for the synthesis rather than it being wasted by enzymes.

A method of not just adding an excess of ATP is adding a substrate fuel which could be Phosphoenolpyruvate (PEP) and Pyruvate Kinase (PK). The substrate PEP acts as a battery which contains the phosphate group. Once the ribosome processes the ATP into an ADP the PK is able to directly collect the phosphate from the PEP and link it back onto the ADP changing it back to an ATP creating a steady loop preserving ATP levels consistent till all the PEP is processed.

Reference List Silverman, A. D., et al. (2020). Nature Reviews Genetics. Caschera, F., & Noireaux, V. (2014). Biological Engineering.

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

Both prokaryotic and eukaryotic cell free systems aim to produce protein from a DNA template. Prokaryotic systems have high speed and yield rates while eukaryotic systems are slower and produce less proteins. In prokaryotic systems the transcription and translation happen in the same place, they are coupled, eukaryotic systems are decoupled and can require extra steps. While the eukaryotic cell free expression is quite costly the prokaryotic based system is inexpensive and simpler to set up and run. However, the eukaryotic system can offer advanced protein folding and more complex modifications as it is able to mimic human-like post-translational modification. The prokaryotic cannot offer natural modifications and creates basic protein folding.

Unless an experiment requires human-like testing I would choose the prokaryotic cell free expression system as it is simpler, less expensive and more prolific. Additionally, if an experiment requires more stability and better control, the fact that prokaryotic systems cannot offer modifications might also make it more predictable and easier to work with.

The INP inaK protein I have been working with would be suitable for prokaryotic cell-free expression as it is a highly repetitive protein which is commonly found in Pseudomonas Syringae, a prokaryote, but if I use an E. coli extract then the codon bias is the same as the native bacteria so the ribosome will read with ease the repetitive portions of the sequence. Moreover, inaK only requires a membrane to anchor to, it does need additional complex compounds. Furthermore, I would aim for a high concentration of inaK for my project in order to produce more ice, which then is more suitable to the high yield of prokaryotic systems.

For a eukaryotic cell-free expression system I would choose a complex human protein such as the tPA, tissue plasminogen activator (a clot-buster), which have more complex modifications and toxins, it would benefit from a more advanced and control protein folding method.

Globally, if a protein has a bacterial origin or has a simple structure and needs to be produced in a large quantity then the prokaryotic systems are more suitable. Eukaryotic systems are useful for complex and human or mammal proteins.

Reference List Zemella, A., et al. (2015). Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. ChemBioChem. Silverman, A. D., et al. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics. Endo, Y., & Sawasaki, T. (2006). A cell-free protein synthesis system for high-throughput proteomics. Journal of Structural and Functional Genomics. (Focusing on Wheat Germ advantages).

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.

I will be using inaK as it is a membrane protein and use a prokaryotic system.

The first issue I will be faced with is that the inaK N-terminal is highly hydrophobic and might create aggregates if I don’t offer it a lipid surface to link to.

I would start with a circular plasmid sourced using Twist and Benchling.

For the expression system I might use an E. coli based S30 extract because the inaK is a bacterial protein and the codon bias and translation speed using the E. coli ribosomes will be naturally optimized for its repetitive sequence. In this case, the transcription and translation will be coupled to ensure the protein folds as it is produced.

I would include PEP and PK for energy regeneration.

Next, as I need to provide the inaK with a new membrane to latch on to I could select a liposome host membrane which might mimic their initial membrane closely, it is structured as small bubbles of phospholipids which should help the first hydrophobic issue.

To determine where exactly to produce the inaK I might have to run a few optimization matrix experiments whether tuning temperature, optimizing lipid to protein ratio to avoid aggregations or a diluted solution, and magnesium and potassium concentrations as membrane proteins are sensitive to charge and a wrong ratio could make the ribosome collapse.

In the end I would have to evaluate the success ratio of my system. The purpose of the inaK is to freeze water, I could place a drop of the cell free solution on a cold plate and observe which solution (supposing I would run a few) would freeze at the highest temperature.

Reference List Hartmann, et al. (2022). Overcoming bottlenecks for in vitro synthesis of ice nucleating protein InaZ. Silverman, A. D., et al. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics. Henrich, et al. (2015). Analyzing the specialized lipid environment of membrane proteins.

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.

A low yield result can be common in CFPS. This can be caused by energy exhaustion, this system is very energy consuming and the PEP might have been consumed too rapidly or the ATP consuming enzymes too active in the sample leading to the ribosome to not have enough energy to produce enough protein. To resolve this a higher concentration of the energy substrate could be used. The DNA template might also be damaged or degraded, causing a blueprint issue. If a linear PCR template is used rather than a plasmid it can happen that enzymes might interfere or damage the ends of the template causing no proteins to be created. Using a controlled plasmid should help avoid this issue, it is possible to make a linear PCR into a circular plasmid through ligations. There might also be an ionic imbalance within the solution which can be due to an unstable ribosome or DNA sequence, this imbalance might work for certain proteins but maybe not the one studied. For instance, mRNA and DNA are very dependent on magnesium and potassium which requires finding the perfect ion balance for the protein in the solution. Adjusting the magnesium ratios can help, this can be done by running multiple tubes with different magnesium percentages.

Reference List Silverman, A. D., et al. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics. Sun, Z. Z., et al. (2013). Linear DNA for rapid prototyping. ACS Synthetic Biology. Caschera, F., & Noireaux, V. (2014). Synthesis of 2.3 mg/ml of GFP. Biological Engineering.

Questions from Kate Adamala

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

1.Pick a function and describe it. a.What would your synthetic cell do? What is the input and what is the output? My synthetic cell design will aim to be used to synthesize inaK (using a prokaryotic cell design) and allow it to more efficiently catalyze ice. Using CFPS for inaK synthesis enables me to control and organise the proteins to sit on the membrane surface in high concentration. Ice nucleation is a surface dependent process and through CFPS I could target the ice nucleation in active sites or clusters which will be more effective than loose individual proteins. The input is what I would give the cell to stay active. I would input a codon optimized plasmid containing the inaK sequence serving as genetic instructions. I would also input the standard set of 20 amino acids in order to build the inaK protein chains. I would input an ATP for energy (with PEP and PK to have energy regeneration). Finally, I would input magnesium and potassium salts to stabilize the internal soup of the cell. If I am designing a prokaryotic cell design then the transcription and translation are coupled and happen simultaneously. The output of this design creates a functional change, primarily the folded inaK proteins anchors to this new membrane and forms an ice active surface. The proteins are physically aligned in a new structure allowing for the crystal lattice of surrounding water molecules. Finally, there would be inorganic phosphate and heat outputs which will be a result of the ATP function.

Reference List Noireaux, V., & Libchaber, A. (2004). A vesicle bioreactor as a step toward an artificial cell. PNAS. Silverman, A. D., et al. (2020). Quarto: A User’s Guide to Cell-Free Protein Synthesis. Nature Reviews Genetics.

b.Could this function be realized by cell-free Tx/Tl alone, without encapsulation? This design could be realized by cell-free Tx/Tl without encapsulation but it will change the function of the synthesis. If I remove the membrane then the inaK depending on it to efficiently catalyze ice would significantly change. The ice nucleation would decrease as I would lose the opportunity to design and arrange clusters but the protein can still technically be synthesized using the 20 amino acids and the DNA template and it would still catalyze ice to some extent. The main restriction will be that the hydrophobic N-terminal will have no membrane to latch onto and it will create disorder aggregates disturbing the ice formation. As much as this synthesis can technically be realized without encapsulation it would not be beneficial or very useful to the experiment.

Reference List Schmid, S., et al. (2016). Probing the ice-nucleation activity of the Pseudomonas syringae InaK protein. (Discussing the importance of membrane context). Noireaux, V., & Libchaber, A. (2004). A vesicle bioreactor as a step toward an artificial cell. PNAS.

c.Could this function be realized by genetically modified natural cell? Yes this function could be realized in a genetically modified natural cell, E. coli was used as a living cell before CFPS became a common technique. Here the inaK could be inserted as plasmid into a lab strain of E. coli to serve as a host. The advantage would be that it is energy self-sustainning as it is living,however, it has a higher complexity rate as there are many other interfering proteins and it has a high bio-safety risk as living GMOs can escape and multiply. A cell free system would need a controlled energy input but has a simpler and safer function. The synthesis efficiency is also much higher and more robust in CFPS than in natural cell.

Reference List In a Natural Cell: The surface of a living E. coli is crowded with lipopolysaccharides (LPS), flagella, and other proteins. These can “get in the way” of the InaK anchors, potentially lowering the density of the ice-active patches. In a Synthetic Cell: You can create a “naked” lipid bilayer. This allows the InaK proteins to pack together tightly without any biological “clutter,” often leading to a more efficient ice-nucleation point.

d.Describe the desired outcome of your synthetic cell operation. Ideally the inaK protein would catalyze ice in a more efficient and organized manner which could be used in glaciers to help preserve and rebuild their natural ice formation and be more resistant to warmer temperatures.

2.Design all components that would need to be part of your synthetic cell. a.What would be the membrane made of? To create a suitable cell and membrane for the inaK protein and its ice nucleation function I would start with a primary lipid mix of 70% POPE (palmitoyl oleoyl sn glycero phosphoethanolamine), helping creating necessary curvature and lateral pressure in membrane thanks to its cone shape, and 30% POPG (palmitoyl oleoyl sn glycero phosphoglycerol), provides negatively charged lipids to help inaK proteins be inserted in the membrane. The common mixture for mimicking an E.coli or Pseudomonas (inaK is commonly found in Pseudomonas Syringae) membrane is composed of PE (phosphatidylethanolamine) and PG (phosphatidylglycerol). This method would prevent aggregation of inaK as the hydrophobic N-terminal cannot insert itself into too stiff of a membrane. Furthermore, the lateral pressure of the membrane will be imitated and this tension helps push the protein together to cluster the inaK and enable ice nucleation at a higher temperature.

Reference List Schmid et al. (2016) found that the ice-nucleation activity of InaK is highly dependent on being embedded in a lipid bilayer rather than just being free-floating, specifically noting that PE/PG mixes provided the most “natural” environment for bacterial anchors.

b.What would you encapsulate inside? Enzymes, small molecules. In order for the cell not to just be a lipid membrane bubble I will add a variety of synthetic cell internal organs which ideally would focus on improving the speed of the transcription and translation system as the inaK is a long and repetitive protein. The cell will encapsulate the genetic blueprint, the DNA plasmid containing the inaK codon optimized gene sequence. Additionally, there should be a strong promoter and double terminator to process large amounts of mRNA better and to ensure the RNA polymerase end in a specific chosen section preventing useless loss of energy. I could add a PURE system to the cell replacing a cell lysate, this is a purified protein synthesis mix including 10S ribosomes (builds the protein), T7 RNA polymerase (turns DNA to mRNA), 36 essential enzymes to charge tRNA and translate information ( aminoacyl, IF,EF,RF) and tRNAs (transfer RNAs moving amino acids to the ribosome). Moreover, I will add the energy source for the PURE system to work. This will include the set of 20 amino acids to build the inaK protein chain, NTPs (ATP, GTP, CTP and UTP) as energy for building the protein sequence and the secondary energy substrate, here creatine phosphate combined with creatine kinase commonly used in PURE systems to keep the ATP charged. To ensure the protein gets to the membrane wall correctly and efficiently I might add SRPs (signal recognition particles) which will connect to the inaK and guide to the membrane and chaperones (DnaK, DnaJ and Grpe) which help with protein folding and prevent the inaK anchor to create clumps before reaching the membrane. Finally, to stabilize the cell I will add magnesium salts.

I used Gemini to help me organize my information.

Reference List Noireaux, V., & Libchaber, A. (2004). A vesicle bioreactor as a step toward an artificial cell. PNAS. This paper provides the foundational protocol for encapsulating T7-based expression systems inside POPE/POPG vesicles. Cui, Y., Chen, X., Wang, Z. and Lu, Y. (2022) ‘Cell-free PURE system: evolution and achievements’, Biodesign Research, 2022, art. no. 9847014. doi: 10.34133/2022/9847014.

c.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) For the inaK CFPS I have chosen a prokaryotic system using an E. coli based PURE method, because it is bacterial the codon language between the Pseudomonas prokaryotic bacteria origin of the inaK and the synthetic prokaryotic cell will match and the ribosome function will be more efficient. The Tx/TI system will then be coupled which works better for a membrane protein and here my hydrophobic anchor will find the membrane immediately avoiding complications. Additionally, in this case a mammalian system might struggle with the repetitiveness of my sequence. Using a prokaryotic E. coli PURE design will give me a higher yield.

Reference List Cui, Y., et al. (2022). ‘Cell-free PURE system: evolution and achievements’, Biodesign Research. This paper confirms that the E. coli-based PURE system is the most effective defined platform for expressing and inserting bacterial membrane proteins.

d.How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?) The cell will communicate mainly with its external environment through the protein action itself, the inaK has an ice nucleating function which will translate as a physical frozen output, the surrounding water molecules will also align and will themselves freeze. Further membrane modifications could also allow for more communication with its environment whether through adding pores to the membrane to create an ATP reactive cell if it detects surrounding ATPs, if not it would stay dormant.

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

inaK: protein derived from Pseudomonas syringae, target gene
T7 RNA polymerase : transcription (if the PURE system doesn’t already contain a protein, the gene is used to produce polymerase to transcribe inaK)
secY, secE, secG : translocation genes
POPE: lipid building blocks
POPG : lipid building block
70S ribosomes: translation
20 amino acids: translation
tRNA: translation
SRP: connects inaK to membrane
hlyA : communication gene (used if wanted to form pores in membrane)

Used Gemini to help me organize my information.

Reference List Cui, Y. et al. (2022) ‘Cell-free PURE system: evolution and achievements’, Biodesign Research. (For the PURE components). Noireaux, V. and Libchaber, A. (2004) ‘A vesicle bioreactor as a step toward an artificial cell’, PNAS. (For the POPE/POPG and $\alpha$HL pore strategy). Li, Q. et al. (2012) ‘Characterization of the ice nucleation protein InaK’, Journal of Biological Chemistry. (For the inaK gene details).

b.How will you measure the function of your system? I could try protein localization through fluorescence protease protection assay using fluorescent tags at the inaK terminals. I could measure the freezing efficiency once again through a droplet trial, testing droplets of multiple versions of the solution when they freeze at what temperature and how resistant they are to temperature.

Homework question from Peter Nguyen

1.Write a one-sentence summary pitch sentence describing your concept. I could use cell free systems adapted to producing inaK in order to directly inoculate glaciers with the aim to preserve and boost ice formation, it would help glaciers rebuild and be more resistant to increasing temperatures caused by climate change. However, I would be interested in pushing the idea of geotextiles already helping preserve glaciers and design a living material, with inaK with a boosted ice nucleation function to create proactive glacier covering actively working to rebuild and preserve glacier ice.

2.How will the idea work, in more detail? Write 3-4 sentences or more. The inaK would be synthesized through a cell free model, using an alternative to E. coli which could resist and be active in sub-zero conditions (to stay active in glaciers), such as Oleispira antarctica a psychrophiles bacteria which has evolved to have specialized ribosomes and enzymes able to remain flexible and functional in a frozen environment. Oleispira antarctica contains unique chaperone (Cpn60 and Cpn10) preventing protein misfolds in frozen temperatures. This cell design would include pores in the membrane so it can stay alive in the textile by having an ATP source of input. These then freeze dried cells would be put into a textile (inoculated during the making of the textile), the textile can be brought to location and installed on the glacier and then be rehydrated to allow ice nucleation of the glacier to begin. This would permit me to create a live material that would be dormant in production and transportation and control its freezing function (preventing the textile from accidentally freezing its surrounding). Note that because I am working with ice nucleation there might be challenges in freeze drying these cells.

Reference List Ferrer, M. et al. (2003) ‘Low temperature-induced systems failure in Escherichia coli: Insights from rescue by cold-adapted chaperones’, Journal of Biological Chemistry. Cui, Y. et al. (2022) ‘Cell-free PURE system: evolution and achievements’, Biodesign Research. D’Amico, S. et al. (2006) ‘Psychrophilic microorganisms: challenges for life’, EMBO reports.

3.What societal challenge or market need will this address? This addresses the environmental, social and political issue of melting glaciers caused by climate change, which only increases the power of climate change as glaciers are key factors in slowing climate change. We are actively losing biodiversities and ecosystems and doing very little about it. It is not seen as a profitable income so little motivation is inputted. However, in the longterm, this irreversible damage done to our nature will actively make climate change worse, and there will be many destructive environmental, social and economic consequences driven by this overlooked issue.

4.How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? Working at very large scale, scale of the glacier, it can be a challenge to efficiently rehydrate the living material as it would be very energy consuming and costly to do it manually, but, if the living geotextile is strategically implemented at the right time of the year (early spring, already when they glacier coverings are usually installed) then nature itself through rain could activate the material naturally. The aim is to limit the human labor impact and simply give nature a tool to reinforce what it already knows how to do. Considering the one time use issue, geotextile coverings which are already used to protect glaciers are removed and installed yearly according to their natural ice melting and forming cycles. The next step of my research would be to find a way to keep the textile created and reabsorb it with new inaK cell free protein systems when it is needed next. The goal is to create a regenerative textile and closed loop system to avoid waste through one time solutions.

Homework question from Ally Huang

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) I am interested in exploring the purpose of ice nucleation in cell free design, freeze dried or not, taking the shape of a multipurpose textile which can be used as an alternative to current voluminous refrigeration tools or a freezing textile to activate. In space stations like the ISS a lot of research relies on lab samples being preserved in sub zero temperatures, from human research samples to organisms or protein crystals. Within research some experiments need cold induced phase changes to be activated or triggered. This technology could also be used for food or medical supplies.

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) The genetic target of this project would be the inaK ice nucleating protein, commonly found in Pseudomonas syringae, with a wide potential of freezing functions.

3.Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) The challenge is optimizing cold packaging and storage systems, a material which would require less space or a material able to be activated once in space again having less constraints in terms of space while travelling. The inaK offers a variety of possibilities in a cell free system whether freeze dried or not as it has a focused and controlled function to freeze. According to the development of the product it can be chosen at what temperature it freezes or activates and how resistant it can be to external temperatures. Creating a highly controlled and bespoke design for certain use in space allows for better control on the research done in space, every aspect of the research can be tailored in hopes to improve success rates of experiments. InaK is a relatively easy INP to work with.

4.Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) I am interested in creating polyvalent designs with multiple usages and applications, this project aims to find an optimal alternative refrigerating system which can have bespoke qualities specific to in space research. As small of a detail it might seem every aspect and tool of experiments impacts the result of research and can lead to better efficiency, results or unexpected breakthroughs. During a space mission all equipment has to be optimized due to lack of space and need for many items and a polyvalent tool that can respond to a wide range of uses can help with the space optimization.

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 design a cell free system for the inaK ice nucleating protein, freeze dry some and then create living textiles, some active and some dormant. The practicality of a textile is that it can be molded, cut, sewn, layered to adapt to any existing object which would then need a freezing function. I can control the amount of inaK for the freezing rate needed, experiment with the different temperatures it can freeze at and the different temperatures it can stay frozen at, I can explore the threshold of the inaK. I would then test the reactivation rates, how much water is needed and how long it would take.