Week 9 HW: Cell Free Systems

The highlighted sections are questions I am unsure how to answer, but made the educated assumptions from the lecture and research.

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.

1. Mutations are still a phenomenon being understood in a synthetic cell system, however, in a cell FREE system, your risk for mutational error is lessened without an entire cell incorporated.
2. Therapies in the environment and on people without the elevated risk of cellular contact
3. The system is overall less time intensive allowing for reactions to happen in a day rather than a multi-day to multi-week turn around time.
4. So far, the cell free system is projected to be less expensive than cell dependent systems as the labor, components and reaction times are less “problematic”.
5. My assumption is this also allows increases the iterative aspects to experimentation. If the system takes less time with less resources and costs, researchers can directly build upon their experiments quicker.

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

Component | Role of the Component

DNA/Genes: Vital! This template specifies the protein of interest to express

Energy Regeneration/ATP: Supplies the energy for the system to do its thing! Protein synthesis is expensive and needs energy ready to be used for your desired amount of time

tRNA: tRNA works with the ribosome to form the amino acid chains (polypeptides) from the mRNA

Cell extract: ribosome, RNA polymerase: needed for transcription and translation

Buffers: Keep the environment optimal for the reaction to occur

NTPs/Amino Acids: amino acids in the system are needed to create the polypeptides that create the desire proteins - Essentially the building blocks of life

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.

Energy provision regeneration is critical because in order for the cell free system to work, it needs energy. Protein synthesis can be an energy hefty system within a cell. However, unlike the cell free system, a cell is continuously out-sourcing food, digesting and, therefore, obtaining more and more energy. A cell-free system does not have that slew of sources that a cell can access energy replenishment, so the researcher or environment has to supply energy via ATP. For continuous ATP supply, the process of phosphorylation is used.

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

Prokaryotic cell-free expression systems: Like its cellular system, it is cheaper, faster and typically yields higher results from the system compared to Eukaryotic systems. I would produce chromo-proteins in a prokaryotic cell-free system because it is typically a singular gene insert and the results can be identified through a visual indicator via its color expression.

Eukaryotic cell-free expression systems: Slower, and more expensive to produce due to the increased complexity of the system that a researcher is attempting to replicate in its broken down parts in a cell free system. However, if you are attempting a more complex pathway, a eukaryotic cell free system is a better option. I would attempt expression of Xylindein, a pigment from the organism Blue Elf Cup due to its pathway complexity.

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 need a lipid bound environment in order to properly fold and maintain structural stability. If you attempt to express membrane proteins in a cell-free system, the proteins could aggregate and mis-fold. Membrane proteins are hydrophobic as they exist inside of the membrane, if they are in water, they precipitate to the top!

I would address these issues by adding liposomes to the mixture. The liposome will encapsulate the membrane protein within its phospholipid structure preserving its structure and function!

“Hydrophobic chemicals associate with the bilayer. This property can be utilized to load liposomes with hydrophobic and/or hydrophilic molecules, a process known as encapsulation.” - Wikipedia

Fun fact: liposomes are used for drug-delivery!

https://en.wikipedia.org/wiki/Liposome

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.

If there was a low yield of my target protein I would increase the energy supply in my solution (the ATP etc) as this would extend the system’s ability to draw energy in for transcription and translation. I would tune the buffer solution to ensure the reaction is happening in a stable environment (pH, salts, trace elements etc). If my template DNA is degrading or not being expressed properly through promoter selection, I would purify my DNA to ensure a pure product and do promoter titer tests to assess the concentration or product yield of different promoters. I would mainly use inducible promoters to regulate concentration and expression without necessarily changing the construct.

1. Pick a function and describe it.

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

My synthetic cell would produce the enzyme urease: an enzyme initiating calcification.

Input: Urease enzymatic pathway.

Output: Calcification of macro and micro cracking that appears in cultural heritage objects like ceramics and stone.

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

I believe it could be realized by a cell-free tx/tl alone without encapsulation in order to get the crystallization product for cultural heritage purposes. However, if studying the origins of life and in understanding the bio-mineralization process in organisms, encapsulation is important as containment allows for specificity in the cells mechanism to crystallize.

https://pubs.acs.org/doi/10.1021/acs.chemrev.5c00659

Could this function be realized by genetically modified natural cell?

Yes and It already is via culturing S.pasteurii, however, the process is laborious and multi-step creating a feasibility complication for art conservators using this system.

Describe the desired outcome of your synthetic cell operation.

The desired outcome would be the crystallization from urease to fill micro and macro cracks in ceramic and stone cultural heritage objects without the added biohazard risk of cellular implementation. It would be a controlled system that mitigates risk and harm of art conservators and the objects themselves.

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

What would be the membrane made of?

The membrane would be made of phospholipids. This will mimic a cellular environment.

What would you encapsulate inside? Enzymes, small molecules.

I would encapsulate the urease enzymatic pathway (the genetic pathway) in a construct, NTPs, cytoplasm, tRNAs, ribosomes, amino acids, communication, and co factors. (This list includes the TX/TL system).

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)

My TX/TL system will come from bacterial systems as the urease pathway is being extracted from S.pastuerii.

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

The synthetic cell will communicate (molecules moving in and out!) through ureI, a membrane protein, which creates a membrane channel for urea to pass in and out without complication.

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: Cholesterol and fatty acids

Genes: ureA, ureB, ureC, ureE.

Tx/Tl system: the bacterial cell free system from the robust species of E.coli

Output product: Bio-mineralized product via calcification

How will you measure the function of your system?

I would measure the function of my system by testing how much calcium carbonate was precipitated and calcified. When briefly researching the direct technology or protocols to follow for assessing calcium carbonate calcification, these were the results:

1. Thermogravimetric analysis
2. Calcium depletion https://pmc.ncbi.nlm.nih.gov/articles/PMC8621315/

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.

Imagine a bandaid that contains a lyophilizied cell-free reaction where the gene is the proteins that ticks use to secrete the anesthetic when they bite. This would allow for immediate pain relief for both small and extreme topical health issues. How will the idea work, in more detail? Write 3-4 sentences or more.

The idea starts with the immediate line of defense found in your at home first-aid kit: the bandaid. By redesigning the patch with pores that contain lyophilizied Tx/Tl system, tick anesthetic genetic pathway, and cell-free maintenance elements (energy regeneration, amino acids etc…), a topical non-invasive pain relieving system is administered. Ticks are incredibly annoying. Beyond having your blood sucked, they are incredibly difficult to notice unless you are frequently checking yourself. Ticks are literally bititng you! Which should be painful! But ticks have developed an evolutionary advantage against their host in which they release a chemical into the local area of the bite, effectively numbing any pain the host may feel for prolonged periods of time. The tickpatch leverages this capability through the cell-free system by allowing it to be shelf-stable and immediately effective when needed.

Through further investigation, tick anesthetic is NON-TOXIC to humans. Some further pluses to the system:

1. Tick proteins have been tested in vivo (allergic asthma studies show HA24 safety)
2. Duration of action is limited to local administration
3. Lyophilized form eliminates risk of live pathogen transmission

What societal challenge or market need will this address?

The societal challenge this addresses is accessible and localized anesthetic treatments. The system would provide pain-relief available at home that are not invasive pain relievers. This could also be used by EMT professionals to treat localized incidents that do not need full anesthetic or expensive alternatives.

How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? The limitation of activation would be overcome by the water content in the blood and by micro-pores imbedded in the bandaid with water to activate the cell free system if blood is not at the site. Bandaids are already a one time use application in order to mitigate contamination risks and isolate the cut.

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/ .

https://www.nasa.gov/hrp/risks/

The webpage on the above was utilized to provide background information on the issues astronauts and space personnel face when going up to space. This helped in the deciding factors of my answers below!

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)

“No current ground-based study exists to test different oxygen exposure levels on humans while they’re experiencing all the stressors of spaceflight.”

Astronauts in space are dealing with a slew of issues pertaining to their health due to the extreme environmental changes the body goes through from earth to space. The risk for mild hypoxia is one of those leering issues - a condition that occurs when oxygen levels in the body become lower than usual. Mild hypoxia creates inconvenient feelings of nausea, confusion and vision impairment.

There are developing solutions to acclimate pressure to oxygen, but I propose a bio-sensor that will monitor oxygen exposure levels during space flight by using the Bio-Bits kit. The significance of this research is relevant to the health concerns that astronauts are facing. This proposal serves as a proof of concept system in identifying possible health shifts in the presence of extreme enviornmental changes. 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)

Hypoxia Inducible Factors (HIF proteins) are inhibited by the cellular machinery typically, however, when oxygen levels are depleted, HIF proteins are expressed and accumulate. These proteins shift the cell from an oxygen environment to an anerobic environment to maintain homeostasis during the oxygen drops.

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

The genetic target of HIF proteins would diagnose possible environments in space of which hypoxia is most likely to occur through the increase of its expression. HIF proteins are only expressed when the body is expressing stress from oxygen depletion. The HIF subunits accumulate and enter the nucleus to complete the complex of the HIF proteins.

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

I hypothesize a HIF protein complex tagging system in which the higher the expression of the HIF protein complex in micro-gravity to macro-gravity environments demonstrate the increased possibility for Hypoxia to occur in astronauts during spaceflight. By doing this research in space and on the ground, researchers can begin identifying how the HIF protein complex changes in response to the extreme environments and additional cellular stressors as a result more specifically. The Bio-Bits would supply the cell-free protein expression kit to do these experiments in the active environment of which this occurs, not just the simulated environment.

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)

Experimental Plan Outline:

All experiments in this set up (besides the outlined control) will be done on the ISS.

1. Use miniPCR to amplify DNA encoding for the HIF Protein Complex within humans.
2. Add the amplified sequence to the BioBits Cell Free System kit.
3. Let the system incubate for 24 hours and induce GFP expression
4. Measure Fluoresence via imageJ
5. Repeat the experiment in and outside the ISS

Control:

On earth, lab personnel will conduct the same experiment in real time to compare expression of the HIF complex within earths gravitational pull.