Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Assignment (Class - Ethics) 1. Describe a biological engineering application or tool you want to develop and why.
Week 2 HW: Read, Write, and Edit DNA
Part 1: Benchling & In-silico Gel Art Part 2: Gel Art - Restriction Digests and Gel Electrophoresis See Week 2 Lab. Part 3: DNA Design Challenge 3.1. Choose your protein.
Post-Lab Questions 1. Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications. Dettinger et al. (2022), “Open-source personal pipetting robots with live-cell incubation and microscopy compatibility,” published in Nature Communications. The authors introduce PHIL (Pipetting Helper Imaging Lid), an open-source, low-cost pipetting robot designed for liquid handling during live-cell experiments and microscopy workflows. PHIL is important because it addresses a real problem in academic labs: many experiments are small-scale, frequently changing, and not well suited to large industrial automation systems, which are often expensive and hard to adapt.
Week 4 HW: Protein Design Part 1
Part A. Conceptual Questions 1. 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) Assuming 500 g of meat contains about 20–25% protein by weight, that piece has roughly: 100–125 g of protein 100 daltons = 100 g/mol so 100–125 g of amino acid residues = about 1.0–1.25 moles 1 mole = 6.022 × 10^23 molecules So the total is about: 6.0 × 1023 to 7.5 × 1023 amino acid molecules 2. Why do humans eat beef but do not become a cow, eat fish but do not become fish?
Week 5 HW: Protein Design Part 2
Part A: SOD1 Binder Peptide Design (From Pranam) Part 1: Generate Binders with PepMLM Sequnce: MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Binder Pseudo Perplexity WRYYVAAVALWX 16.2395343215211 WHYYAVAAEWKX 13.6945052943038 WLVPAAAAAHGK 7.93406338297721 WRYGPVAVRHWK 14.3797355975152 FLYRWLPSRRGG 20.6352312728361 Part 2: Evaluate Binders with AlphaFold3 “PepMLM outputs X; substituted X→A for AlphaFold input.” Binder ipTM Score WRYYVAAVALWA 0.34 WHYYAVAAEWKA 0.23 WLVPAAAAAHGK 0.48 WRYGPVAVRHWK 0.36 FLYRWLPSRRGG 0.38 Across the five AlphaFold3 complex predictions, ipTM values ranged from 0.23 to 0.48. The known binder FLYRWLPSRRGG gave ipTM = 0.38 and appeared weakly defined, remaining largely surface-adjacent/partially detached rather than buried in a clear pocket, with no obvious localization near the N-terminus where A4V sits. Three PepMLM peptides (WHYYAVAAEWKA, 0.23; WRYYVAAVALWA, 0.34; WRYGPVAVRHWK, 0.36) similarly showed low-confidence interfaces, tending to lie loosely on the β-barrel exterior instead of concentrating at the A4V region. In contrast, WLVPAAAAAHGK produced the strongest prediction (ipTM = 0.48) and appeared more plausibly docked along a β-barrel/loop-adjacent surface, making it the only PepMLM-generated peptide that exceeded the known binder’s ipTM in this set. Part 3: Evaluate Properties of Generated Peptides in the PeptiVerse
Week 6 HW: Genetic Circuits Part 1
Part A: DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion High-Fidelity PCR Master Mix is a 2X mix containing Phusion DNA Polymerase, nucleotides, and an optimized reaction buffer including MgCl₂. Functionally, the polymerase synthesizes the new DNA strand, the dNTPs are the nucleotide building blocks it incorporates, the buffer maintains the reaction chemistry, and Mg²⁺ is the essential cofactor that enables polymerase activity and helps stabilize primer-template interactions. Phusion is called “high fidelity” because it has 3′→5′ exonuclease proofreading activity, which lowers the error rate compared with standard Taq-type PCR enzymes. What are some factors that determine primer annealing temperature during PCR?
Week 7 HW: Genetic Circuits Part 2
Assignment Part 1: Intracellular Artificial Neural Networks What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs have an advantage over traditional Boolean genetic circuits because they can respond to graded, noisy biological signals instead of forcing every input into a simple ON/OFF state. This makes them better for tasks like classification, where a cell needs to weigh several signals together and make a more flexible decision, while Boolean circuits are better for simpler yes/no logic. Describe a useful application for an IANN; include a detailed description of input/output behavior, as well as any limitations an IANN might face to achieve your goal.
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. Cell-free protein synthesis is more flexible than in vivo expression because you can directly control the reaction conditions without keeping cells alive. It is especially useful for making toxic proteins and proteins that are hard to express in living cells, like some membrane proteins. Describe the main components of a cell-free expression system and explain the role of each component.
Week 10 HW: Imaging and Measurement
Homework: Final Project For the final project, I will primarily measure whether my designed DNA templates are correct and whether they can produce proteins in a T7 cell-free expression system. I will verify the DNA constructs using DNA sequencing and agarose gel electrophoresis, then test expression using two templates: a minimal ureABC structural urease construct and a separate sfGFP reporter construct. The sfGFP template will serve as a visual positive control, and its expression will be measured by fluorescence, while expression of UreA, UreB, and UreC will be measured using SDS-PAGE. I may also perform an exploratory urease-related activity assay, but this is not the main measurement because the minimal structural construct does not include the full accessory genes required for strong urease activation. Homework: Waters Part I — Molecular Weight Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight?
Part B: Cell-Free Protein Synthesis | Cell-Free Reagents Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction. BL21 (DE3) Star lysate (includes T7 RNA polymerase): This provides the core cellular machinery needed for protein synthesis, incl~uding ribosomes, tRNAs, enzymes, and other translation factors. Because it includes T7 RNA polymerase, it can also transcribe DNA templates with a T7 promoter into mRNA.