Homework Part A

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory for Committed Listeners and MIT/Harvard Students

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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links:

https://dnadots.minipcr.com/wp-content/uploads/2019/09/DNAdots-Cell-Free-Tech-final_qnoa.pdf

https://pubs.acs.org/doi/10.1021/acssynbio.3c00733

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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.

   Ans: Cell-free protein synthesis (CFPS) offers superior flexibility and control compared to traditional in vivo methods. Key advantages include:

   • Open-system manipulation: Researchers can directly add non-canonical amino acids, isotopic labels, or folding chaperones to reactions
   • Toxic protein production: CFPS avoids cell viability issues, enabling synthesis of proteins lethal to living hosts
   • Rapid prototyping: Reactions bypass cloning and cell culture steps, reducing setup time from weeks to hours

   Cases favoring CFPS:

   1. Membrane proteins: CFPS allows co-translational insertion into supplied liposomes or nanodiscs, improving solubility and folding
   • High-throughput screening: Parallel synthesis of multiple variants (e.g., mutagenesis libraries) without cloning

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

   Ans: A CFPS system requires:

   • Cell lysate: Contains ribosomes, tRNA, and enzymes for transcription/translation (e.g., E. coli or wheat germ extracts)
   • Genetic template: DNA or mRNA encoding the target protein, often under a T7 promoter for prokaryotic systems
   • Energy substrates: ATP/GTP and regeneration systems (e.g., phosphoenolpyruvate or glucose
   • Amino acids: 20 standard amino acids, often supplemented with isotopically labeled or modified variants
   • Cofactors: Mg²⁺, K⁺, and buffers to maintain optimal pH and ionic conditions

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.

   Ans: ATP depletion rapidly halts protein synthesis. Methods for sustained ATP supply:

   • Glucose-phosphate system: Glucose metabolism regenerates ATP via glycolysis, while phosphate buffers prevent inhibitory inorganic phosphate accumulatio
   • Cytomim system: Mimics E. coli metabolism using glycolytic intermediates (e.g., maltodextrin) for prolonged ATP production

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

   Ans: Prokaryotic vs. Eukaryotic CFPS Systems

   Aspect	Prokaryotic (E. coli)	Eukaryotic (Wheat Germ/Insect)
   Yield	High (1–2 mg/mL)	Lower (0.1–0.5 mg/mL)
   PTMs	None	Glycosylation, disulfide bonds
   Cost	Low	Higher

   Example applications:

   • Prokaryotic: Expressing a bacterial enzyme (e.g., Taq polymerase) requiring no PTM
   • Eukaryotic: Producing a human glycoprotein (e.g., monoclonal antibody) needing mammalian-like glycosylation

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.

   Ans: Design considerations:

   • Hydrophobic environment: Add liposomes, nanodiscs, or detergents (e.g., Brij-35) during synthesis to solubilize membrane protein
   • Chaperones: Supplement with DnaK/DnaJ or GroEL/GroES to aid folding
   • Lysate choice: Use insect or wheat germ extracts with endogenous microsomes for co-translational insertio

   Challenges:

   • Aggregation: Address by lowering reaction temperature (16–25°C) to slow translation
   • Low solubility: Use nanodiscs to stabilize proteins post-synthesis

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.

   Ans:

   Cause	Solution
   RNase contamination	Add RNase inhibitors; purify template DNA
   Codon bias	Optimize codon usage or lower reaction temperature to 30°C
   Energy depletion	Switch to glucose-phosphate energy system

   For template design issues, verify promoter/UTR sequences and include T7 terminators to stabilize mRNA

Homework Part B - Individual Final Project Report

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory for Committed Listeners and MIT/Harvard Students. We’d like students to start exploring their final project in depth this week! The minimum requirement is filling out Aim 1

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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links (For MIT/Harvard): https://docs.google.com/spreadsheets/d/190gXDB_T9lq6wPN_0yLEAU66tPPJehVL1PH5dQZyFAU/edit?gid=70407915#gid=70407915

Key Due Dates (For MIT/Harvard):

• Twist: 4/11/25
• Thermo Fisher: 4/11/25
• AddGene plasmids: 4/11/25
• NEB: 5/1/25 (Rolling Orders)
• GeneWiz: 5/1/2025 </aside>

<aside> 💡 See past projects here: Google Slide Deck.

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<aside> 💡 Publish a copy of this onto on your own website.

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• See this link if you want examples of what you can include in your answers for each question. Note: the question numbers do not line up, and you can ignore any word requirements in this linked document.
• Please include your answers to the following questions on the “final project” page of your HTG(A)A website.

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

About 150 words.

Ans: Living Memory is a digital-into-DNA storage pipeline that simulates how digital files (text, images, or binary data) can be stored in synthetic DNA. The system compresses data, encrypts it using password-based AES, applies Reed-Solomon error correction, and finally encodes the result into a DNA sequence using a 2-bit mapping (00 → A, etc.). Biological start and stop codons are added for contextual framing. While this project was developed entirely in silico, it is designed for real-world implementation via DNA synthesis or integration into living organisms. This platform proposes a post-silicon paradigm where biology is not just alive, but archival. Future extensions could embed this data in cells, simulate mutational drift, and explore searchability via barcoded DNA. The result is a secure, modular, and extensible system that reimagines how humanity stores information through the lens of synthetic biology.

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