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.
1. A couple of the main advantages of using a cell-free system include having no cellular constraints on what you can produce (not limited by the biology of a cell), and the fact that you can take a more focused engineering approach- you can employ rapid design-build-test systems, like programming. Because you are simplifying the expression of a specific protein/suite of proteins, you may lose stochastic information from biology, but you can more narrowly manage what that specific output is. Contexts where using CFPS makes more sense than using normal cellular expression include toxic protein production that would kill the host cells, especially at manufacturing-like volumes, and tuning the specific system to output something that is not natural. When you are creating something that is not found in nature, you may require inputs that are also not found in nature- like unnatural amino acids, such as UAA.
2. To be noted is the cost of CFPS- because my final project outputs toxic venoms which hurt cells, I looked at CFPS. It is 100–500× less yield, ~1,000× higher cost per mg, but would take hours instead of weeks to do the experiment (assuming I can readily access the inputs). Over the last 20 years, however, the costs have dropped 10x every decade, so CFPS is also following an exponential cost drop of 100x over 20 years. Yield output rates increase 3-10x over 20 years.
2. Describe the main components of a cell-free expression system and explain the role of each component.
1. Cell extract: contains ribosomes, tRNA’s, polymerases, enzymes for transcription/translation
2. Energy system: a set of molecules that regenerate ATP/GTP so that transcription and translation can run continuously
3. DNA template: plasmid or linear DNA encoding of the protein you are interested in
4. Amino acids + cofactors: raw materials + helpers for making proteins
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.
1. Transcription and translation use a lot of energy, so they burn through ATP/GTP quickly. ATP/GTP are in high demand, and each system firing needs to have a replenishment, or the forward chains of these fail.
1. Other uses of energy: metabolic pathway prototyping, bio-sensing, CRISPR reactions, anything that uses energy to achieve some outcome
2. Maltodextrin-based energy regeneration:
1. add maltodextrin + enzymes
2. enzymes convert maltodextrin → glucose-1P → ATP (via glycolysis-like pathways)
3. generates ATP slowly and steadily, ideal for long reactions
3. Other methods: PEP system (fast, but short-lived and expensive), creatine phosphate + creatine kinase (clean byproducts), acetyl phosphate
4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

Prokaryotic cell-free expression system	Eukaryotic cell-free expression system
fast, high yield, cheap	slower, lower yield, more expensive
easy to extract and prep	support complex folding, PTMs, disulfide bonds
limited in post-translational modifications	better for human-like proteins, membrane proteins
good for simple proteins

1. I would like to use the proteins from my final project, which include the natural/synthetic snake antivenom and bee melittin, as well as binders. The table is output from ChatGPT.

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.
1. poor transcription or translation initiation, caused by weak promoter, the wrong RBS, or the wrong mRNA structure
1. use strong T7 promoter + ribosomal binding site
2. check mRNA secondary structure near start codon (minimize hairpins, where the RNA folds back on itself, which blocks the ribosome from binding)
3. codon-optimize DNA (rewrite the gene using codons that the host has lots of tRNAs for)
2. protein degradation, caused by endogenous proteases in the extract that are chopping your target protein
1. add protease inhibitor
2. use truncated or tag-protected versions
3. try extracts from protease-deficient strains
3. misfolding/aggregation, caused by complexity of product structure, lack of disulfides, or no chaperones
1. add more chaperones (DnaK, GroEL/ES)
1. these are enzymes, so they lower the activation energy
2. lower reaction temp (20-25C)
3. add folding enhancers (glutathione for disulfides)

Homework Part B - Individual Final Project Report

1. Provide an abstract/narrative/summary for your final project.

This project is based on the paper De novo designed proteins neutralize lethal snake venom toxins. The protocol is designed around a BSL-1 lab and includes an additional experiment for designing bee antivenom using the same computational + expression system. For the snake venom, we are testing if a synthetic protein binder (SHRT) can neutralize a synthetic toxin (ScNtx for snake) and protect human cells. To do this, we will express, purify, and validate SHRT (short-chain α-neurotoxin binder) binding to ScNtx (synthetic short-chain α-neurotoxin consensus), using BSL-1 compatible tools. We are then going to follow the same protocol for Melittin (bee venom), as a way of expanding the current research scope.

2. Provide a background and motivation section for your final project.

a. Explain your motivation for pursuing your project

I originally chose the project because it would take me through the end to end process of designing a drug, using all the new tools. I only realized that we could do bee venom (which I am allergic to) after breaking down the whole paper into an experimental protocol.

b. Describing the current state of knowledge related to your project. Try to cite at least 2 peer-reviewed research papers.

c. Describe how your project is innovative and expand upon the significance of your final project.

We take the systems discussed one step further than the paper, by designing a new binder for a different venom, but the point is to prove it as a repeatable system, like a refinery. The input is someone’s entire digital representation- the output is custom binders on demand, then any chemical that helps manage entropy/harmony for health. For distribution, we would ideally leverage the freeze-dried expression system from the Wyss Lab, meaning you can ship it internationally on drones, or whatever is the fastest form factor. Now, we have an entirely dry end-to-end, custom expression system, to produce any binder from scratch (leveraging computation + sensors + automated distribution chains to do most of the heavy lifting). This then pushes the demand to detection of the venom, where sensing will undergo a massive revolution, and now you have the infrastructure to create a real-time, compile-time, detection-time, end-to-end system of producing any drug in seconds. Constraints outside of silico would, again, be detection and distribution. It becomes an engineering optimization problem rather than a scientific process. :D

3. Aims

For each aim:

1. Create an experimental plan for your final project by including specific methods/tools/technologies/biological concepts for each experiment/analysis steps
2. Present any results/validation of (a part of) your final project. Share what worked, what didn’t work, and why!

1. create an end to end automated pipeline for creating antibodies to bind to antigens
1. Using this general workflow:
1. Get target structure → Use PDB (e.g. Melittin = 2MLT) or predict if not available
2. Pick binding site → Likely the toxic active region of Melittin
3. Generate binder shape → Use a design tool to place a binder near that site
4. Design binder sequence → Convert binder backbone to sequence
5. Validate binding → Predict binder-Melittin complex and check affinity
6. Visualize → Confirm it binds in a useful way (blocks toxicity)
2. express the natural and synthetic venoms and binders in E Coli
3. get the correct synthetic Melittin antibody to actually bind using the software end to end

4. Describe any unexpected challenge(s) you faced in planning or executing your final project.

The amount of words I did not understand and now do understand. I read Engineering Genetic Circuits, Molecular Biology of the Cell, A Genetic Switch, Lambda Phage (Mark Ptashne), Mammalian Synthetic Biology, Synthetic Biology: A Primer, Metabolic Engineering, and countless papers. I reread the antivenom paper at least 4 times, each time taking new notes on a fresh copy.

5. Describe the bioethical considerations involved in your project.

This is a scale business- if you project to the the extreme asymptote, this means that every combinatorial combination of chemical can be made automatically from end to end, only bound by computation; like a serverless function. Both good and bad, and will push defensive capabilities to be in extremely high demand.

6. Describe any future work

Expand this to all chemicals- create a factory, pair with consumer trend + interface, expand. Personal chemicals 24/7 to manage entropy.