Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
1)First, describe a biological engineering application or tool you want to develop and why.( This could be inspired by an idea for your HTGAA class project and/or something for which you are already doing in your research, or something you are just curious about.)
Week 2: DNA Read, Write and Edit
Part 1: Benchling & In-silico Gel Art First I start to stimulate Restriction Enzyme in Lambda DNA: Also shown above,with Ronan’s website, I try to pattern with enzymes. Part 3: DNA Design Challenge 3.1. Choose your protein: I decided to choose GFP protein in water jellyfish(Also called “Aqua Victoria”). Because with GFP protein I can see that organization of cell. I can track protein and find to where to go in the cell. On the shown below, you can see GFP Protein Sequence:
Review this week’s recitation and this week’s lab for details on the Opentrons and programming it. This week was all about moving from manual pipetting to the world of liquid handling automation. I’ve been diving deep into the Opentrons ecosystem, specifically focusing on how to bridge the gap between writing Python code and seeing the robot actually execute those movements on the deck.
Part A. Conceptual Questions How many molecules of amino acids do you take with a piece of 500 grams of meat? Meat is approximately 20% protein by mass. So, 500g of meat contains about 100g of protein. Given that the average mass of an amino acid is 100 Daltons ($1.66 \times 10^{-22}$ grams), we can calculate the total number of molecules:$100g / (100 \times 1.66 \times 10^{-24}g) \approx 6 \times 10^{24}$ amino acid molecules. That is roughly 10 moles. Why do humans eat beef but do not become a cow, eat fish but do not become fish? This is because our digestive system breaks down the proteins we eat into their individual building blocks: amino acids. Our body doesn’t use the cow or fish proteins directly; it uses these free amino acids to assemble “human” proteins based on the instructions in our own DNA. Why are there only 20 natural amino acids? This is often described as a “frozen accident” in evolution. While there are many more possible amino acids, these 20 provided enough chemical diversity (acidic, basic, hydrophobic, etc.) to build complex 3D structures and catalyze reactions. Once life became complex, changing this fundamental toolkit would have been too disruptive. Can you make other non-natural amino acids? Design some new amino acids.Yes, scientists can synthesize non-natural amino acids (ncAAs) by adding unique side chains. For my project on schizophrenia, I could design an amino acid with a fluorescent “sensor” side chain that changes color when it interacts with high concentrations of dopamine. This would allow us to visualize “dopamine storms” in real-time. Where did amino acids come from before enzymes that make them, and before life started? Amino acids likely formed through abiotic synthesis. The Miller-Urey experiment showed that early Earth’s atmospheric gases (methane, ammonia, water vapor) could react with electrical discharges (lightning) to create amino acids. They might have also been delivered to Earth via meteorites. Why are most molecular helices right-handed? This is due to the “chirality” of L-amino acids. Because all life uses L-amino acids, the right-handed $\alpha$-helix is the most energetically stable conformation that avoids steric clashes (physical bumping) between the side chains. Why do $\beta$-sheets tend to aggregate? What is the driving force? The main driving forces are hydrogen bonding and the hydrophobic effect. The edges of a $\beta$-sheet have “unsatisfied” hydrogen bond donors and acceptors. This makes them very “sticky,” leading them to stack with other $\beta$-sheets to achieve a lower energy state. Why do many amyloid diseases form $\beta$-sheets? Can you use them as materials? Amyloid $\beta$-sheets are incredibly stable and resistant to degradation. In diseases like Alzheimer’s, proteins misfold into this “energy well” from which they cannot escape.As materials: Yes! Amyloid fibers are stronger than steel for their size. They can be engineered as ultra-stable nanowires or drug-delivery scaffolds. Design a $\beta$-sheet motif that forms a well-ordered structure. A well-ordered motif can be designed using an “alternating” pattern: (Val-Lys-Val-Glu)n. In this sequence, the hydrophobic Valine residues face one side while the charged Lysine and Glutamate residues face the other. This creates a “Janus-faced” sheet that is oily on one side and water-loving on the other, allowing it to assemble into perfect layers. Part B: Protein Analysis and Visualization 1. Briefly describe the protein you selected and why you selected it. I want to select DRD2(the Human Dopamine D2 Receptor) protein because this protein provided to enter the doapamin in the cell. Also ın neurodejenaretif diseases such as in schzophrenia this receptor protein is overexpressing therefore dopamin enter the cell more than normal and this leads to hallucination. Therefore this protein plays important role in brain.
Week 5 — Protein Design Part II
Part A: SOD1 Binder Peptide Design : First, we change Alanine(A) to Valine(V) at residue 4 in SOD1 sequence. Part 1: Generate Binders with PepMLM: I generate 4 different peptides by using PepMLM Collab. In protein design (ProteinMPNN), Perplexity measures the model’s “uncertainty” when choosing amino acids for a specific position. It indicates how well a designed sequence fits the target protein’s structural constraints.The lower the score (e.g., < 10), the more confident the model is. It means the amino acid sequence is physically and energetically highly compatible with the protein structure. That way we can say that first binder is the most optional for us.
Genetic Circuits Part I: Assembly Technologies
Assignment: DNA Assembly Answer these questions about the protocol in this week’s lab: What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The Phusion Master Mix is a convenient 2X concentrated solution designed for high-performance PCR. Key components include: Phusion DNA Polymerase: A high-fidelity enzyme with a processivity-enhancing domain, ensuring extremely low error rates and fast extension times. dNTPs: The essential building blocks (dATP, dCTP, dGTP, dTTP) required for new DNA strand synthesis. Reaction Buffer: Maintains the optimal pH and provides necessary ions.$ MgCl2: Acts as a vital cofactor for the polymerase enzyme activity. What are some factors that determine primer annealing temperature during PCR? The Ta is critical for primer specificity and yield. It is primarily determined by:
General homework questions 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 primary advantage of cell-free systems is the removal of the cell membrane, which eliminates the “black box” nature of cellular metabolism.
Advanced Imaging & Measurement Technology
Final Project Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc. For my “Sentinel Microbes” project, which aims to detect foreign pathogens in the gut, the most critical aspect to measure is the quantitative expression of the reporter protein. When the engineered microbes encounter a specific pathogen (like Salmonella), they trigger a genetic circuit to produce a visual signal. I need to measure the concentration and identity of this protein to confirm that the “Detected!” signal is accurate and strong enough to be noticed.
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork Part B: Cell-Free Protein Synthesis | Cell-Free Reagents E.Coli Lysate: It is acted as the catalytic core of the reaction; it provides the ribosomes for translation and T7 RNA Polymerase for high-level transcription. Salts and Buffers: Potassium Glutamate: It provides necessary ionic strength and potassium ions for protein folding without inhibiting DNA-protein interactions like chloride salts might. HEPES-KOH pH 7.5: It is pH indicator.Also it prevents reaction from becoming too acidic due to metabolic byproducts. Magnesium Glutamate: It supplies magnesium ions required for stabilizing ribosome assembly and acting as a cofactor for RNA polymerase. Potassium Phosphate (Monobasic- Dibasic): It provides secondary buffering and source of inorganic phosphate needed to regenerate ATP. Energy and Nucleotide System: Ribosome Guanine: It serves as specific region for nitrogenous base that can be salvaged by lysate enzymes to create GTP for transcription when direct GMP levels are low. Translation Mix (Amino Acids): they are provide fundamental building blocks for synthesizing the polypeptide chain.