Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
The Biological Engineering Tool Tool: Portable Cell-Free Allergen Biosensor. Description: A single-use, portable reaction unit containing shelf-stable biological sensing reagents. Mechanism: The user introduces a small sample of food (solid or liquid) into the unit. The device initiates a biochemical reaction that specifically recognizes the molecular signature of a target allergen (e.g., peanut or soy). If the target is detected, the device triggers a distinct visual signal (such as a color change or fluorescence) within minutes.
Week 2 HW: DNA Read Write and Edit
DNA Design Challenge Chosen Protein: I chose GFP because it serves as a robust reporter that could be used for my allergen biosensor. The goal of the device is to turn a biological detection (sensing peanut DNA) into a signal the user can see. GFP spontaneously fluoresces green when exposed to UV or blue light (like a simple black light LED). By designing the system so that GFP is activated only when the allergen is detected (or shut off in the presence of the allergen), I can create an intuitive user interface.
Project Overview: Cell-Free Allergen Biosensor I am hoping to develop a rapid, consumer-grade biosensor designed to detect trace allergens like peanut or soy in a restaurant setting. To prioritize speed and accuracy, I will use a DNA-to-RNA detection circuit. The workflow consists of three main stages: Extraction and Amplification: I could use RPA (Recombinase Polymerase Amplification) to exponentially copy target DNA (like the Ara h 1 gene) at a constant 37°C. Transcription: T7 RNA polymerase can concurrently convert that DNA into Trigger RNA. RNA Toehold Detection: This Trigger RNA can bind to a synthetic Toehold Switch, and unzip an RNA hairpin to allow the translation of a reporter protein. This can create a visible color change or induce luminescence in under 20 minutes. By using a cell-free protein synthesis system, the entire reaction is shelf-stable and functions without the need for a traditional lab environment.
Week 4 HW: Protein Design part I
HW Questions: How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons) Meat is roughly 20% protein by mass, so there’s ~100g of protein in 500g of meat. Average amino acid molecular weight is ~110 Da. 100 g ÷ 110 g/mol ≈ 0.91 mol of amino acid residues × Avogadro’s number: 0.91 × 6.022 × 10²³ ≈ ~5.5 × 10²³ amino acid residues Why do humans eat beef but do not become a cow, eat fish but do not become fish?
Week 5 HW: Protein Design part II
Part A: SOD1 Binder Peptide Design sequences, scores, structure, and properties for all peptides PepMLM binder generation Perplexity scores for known and generated peptides: Alphafold binder evaluation ipTM Values and Comparison to Known Binder The ipTM values across all peptides are low, ranging from 0.27 to 0.43, and none exceed 0.5 — the general threshold for confident protein-peptide interaction prediction. Notably, two PepMLM-generated peptides (Sequence_0 at 0.40 and Sequence_1 at 0.43) actually exceed the known binder (Sequence_4 at 0.32), suggesting the model produced candidates with comparable or slightly better predicted interface confidence. However, all predictions share the same binding
HW Questions What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion DNA Polymease, which synthesized new DNA by adding new nucleotides to the template DNA during PCR dNTPs, which are the nucleotide building blocks (dATP, dGTP, dCTP, and dTTP) reaction buffer, which acts as a chemical stabilizer that maintains the ideal pH and salt balance so the enzyme stays active and can accurately build new DNA strands. What are some factors that determine primer annealing temperature during PCR? Melting temperature of the primer, which is the temperature at which half of the DNA complex dissociates Primer length, since longer primers usually require higher annealing temperatures GC content, since higher GC content typically increases the primer melting temperature Salt concentration, since higher salt concentrations can stabilize the DNA and thus may require higher annealing temperatures There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other. Feature PCR (Polymerase Chain Reaction) Restriction Enzyme Digest Mechanism Enzymatic Synthesis: Building new strands from primers. Enzymatic Cleavage: Cutting phosphodiester bonds at specific sites. Protocol Thermal Cycling: Repeated steps of denaturation (95°C), annealing (55-65°C), and extension (72°C). Isothermal Incubation: DNA and enzymes are mixed in a buffer and held at a constant temp (usually 37°C). Reagents DNA template, Primers, dNTPs, Taq Polymerase, MgCl2, Buffer. DNA template, Restriction Enzymes, specific BSA/Salt Buffer, Water. Pros High sensitivity; amplifies DNA; creates specific fragments without needing existing cut sites. Simple setup; highly reproducible; great for verifying known sequences or circular DNA. Cons Prone to contamination; requires known flanking sequences; potential for polymerase errors. Does not amplify DNA; limited by the location of natural recognition sites. When to Use When you have minimal DNA, need a custom fragment, or want to add “tails” for cloning. When linearizing plasmids, performing diagnostic checks, or subcloning existing inserts. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Both the PCR and digested fragments must share identical overlapping terminal sequences (15–40 bp) with their neighboring fragments to allow for seamless homology-directed assembly. How does the plasmid DNA enter the E. coli cells during transformation? Membrane pores open due to a thermal pressure imbalance during the heat shock, allowing the plasmid DNA (which has been neutralized by calcium ions) to be pulled into the cell. Describe another assembly method in detail (such as Golden Gate Assembly) Golden Gate Assembly is a highly efficient “one-pot” cloning method that allows you to join multiple DNA fragments together simultaneously using Type IIS restriction enzymes and T4 DNA ligase. Unlike standard enzymes, Type IIS enzymes like BsaI bind to a specific recognition sequence but cut the DNA several nucleotides away, creating custom 4-base overhangs. By strategically designing these overhangs to be complementary, you can ensure that multiple fragments assemble in a specific, directional order. During the reaction, you cycle the temperature to repeatedly cut and ligate the DNA until the fragments are perfectly joined. A key advantage is that the enzyme’s recognition sites are positioned to be “cut off” and removed during the process, meaning the final product cannot be re-cut. This makes the reaction irreversible and drives the assembly toward the final, seamless circular plasmid. Because of this precision, Golden Gate is the gold standard for modular cloning and building complex multi-gene constructs. Simulating Golden Gate using AddGene’s tool
week 7 HW: genetic circuits part II
Part 1: Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? Traditional genetic circuits can only read a signal as ON/OFF, even though molecules inside a cell exist at all kinds of intermediate concentrations. To build something complex out of ON/OFF switches, you have to layer many of them together, and each added layer introduces new opportunities for components to accidentally influence each other or fall out of sync. IANNs instead pass graded responses between nodes. Each node receives an actual concentration value, weighs it, and passes a continuous output forward. This means a single node carries far more information than an ON/OFF switch, so you need fewer of them to represent something complex, and there are fewer points at which things can go wrong.