Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
About my project. Question 1 First, describe a biological engineering application or tool you want to develop and why. I’d Like to Do I would like to develop an anthocyanin-based reporter system for Solanum aethiopicus. I would like it to use native genetic elements to fit in with my desire to do ethical biotechnology that fits in my own beliefs.
Week 2 HW: DNA Read Write and Edit
Part 1 Part 2 Gel Art Part 3 3.1. Choose your protein.
My Design on https://opentrons-art.rcdonovan.com/ My Design on Opentrons Colab My Opentrons instructions from opentrons import types
Week 4 HW: Protein Design Part 1
PART A Conceptual 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) Assuming ~20–25% protein in meat: 500 g meat → 100–125 g protein With average amino acid mass = 100 Da ≈ 100 g/mol: that protein corresponds to 1.0–1.25 moles of amino acids, i.e. (6.0–7.5) × 10²³ amino acid molecules.
Week 5 HW: Protein Design Part 2
Part 1: Generate Binders with PepMLM Sequence Retrieval MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Mutated with A4V MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Binder Pseudo Perplexity Seq1 HHVPVVVLRHKX 16.326572 Seq2 WRYYAAVARWKE 13.897447 Seq3 HRYYPAAARWKX 8.531565 Control FLYRWLPSRRGG 20.63523127283615 Part 2: Evaluate Binders with AlphaFold3 Navigate to the AlphaFold Server: alphafoldserver.com For each peptide, submit the mutant SOD1 sequence followed by the peptide sequence as separate chains to model the protein-peptide complex. Record the ipTM score and briefly describe where the peptide appears to bind. Does it localize near the N-terminus where A4V sits? Does it engage the β-barrel region or approach the dimer interface? Does it appear surface-bound or partially buried? In a short paragraph, describe the ipTM values you observe and whether any PepMLM-generated peptide matches or exceeds the known binder. Sequence 1 HHVPVVVLRHKX The sequence was submitted without the last X because it was flagged as an illegal character (it could stand for various amino acids). The peptide appears to be interacting with ipTM = 0.22 pTM = 0.85 pTM = 0.85 suggests the overall fold is very reliable, while ipTM = 0.22 says the predicted interaction/interface between chains is very poor. This means the protein’s global shape looks strong and likely meaningful, this is in line with it being a known and well illucidated human protein. It seems like a case where AlphaFold is confident about folding, but not about binding geometry.
Week 6 HW: Genetic Circuit Part 1
What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion DNA Polymerase - this is for amplifying the DNA by polymerisation. It is special because when used with its special buffer, it has up to 52× higher fidelity rate than ordinary Taq polymerase dNTPs- These are the building blocks that are used in the polymersiation by the DNA polymerase MgCl₂ - DNA polymerase enzyme needs magnesium ions to function properly (they are necessary cofactors for the thermostable enzyme) Optimized buffer - Phusion needs an optimized buffer because it uses a highly processive and high-fidelity DNA polymerase (originally a Pfu-like enzyme fused to a processivity domain) that is very sensitive to Mg²⁺, ionic strength, pH, and additives. Therefore these components must be balanced perfectly for the PCR not to fail in terms of processivity or fidelity. These items are added to the PCR: template DNA, primers, and molecular water. What are some factors that determine primer annealing temperature during PCR? Here’s the table simplified into bullet points:
Week 7 HW: Genetic Circuit Part 2
Advantages of IANNs over Boolean genetic circuits Traditional genetic circuits (AND/OR/NOT) are great when the world can be cleanly thresholded into ON/OFF inputs and you only need discrete outputs. IANNs (genetic implementations of artificial neural networks) are useful when biology is messy—inputs are continuous, noisy, and correlated Analog, not just digital IANNs naturally support graded responses (continuous input → continuous output), not only Boolean truth tables. That lets you encode “how much” and “how confident,” not just “yes/no.”
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 can be used even when cold-chain conditions cannot be assured -Cell-free systems can be used without cloning and creating recombinant organisms Cell-free protein synthesis offers flexibility and a lot of control because you can directly tune reaction components (DNA amount, salts/cofactors, energy mix, chaperones, redox environment) and test many conditions rapidly without cell growth, regulation, or viability constraints. Cell-free expression is especially beneficial for rapid prototyping of constructs/circuits and also producing products especially if they are tocix to living expression systems. It can also enable on-demand expression (e.g., freeze-dried systems) and reactions requiring tightly controlled chemistry. This is great for making vaccines on demand on a mission to Mars for example. Describe the main components of a cell-free expression system and explain the role of each component. For General Systems Cell extract (lysate) or purified translation system — supplies the machinery that makes protein (ribosomes, tRNAs, translation factors, enzymes).
Week 10 HW: Imaging and Measurement
For your 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. I intend to measure the presence of cyclotide activity using the GFP signal from a reporter construct. I also intend to measure the extent of membrane lysis caused by the presence of my cyclotides. Controls to make sure the system is working. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. A. eGFP reporter signal (cyclotide activity screen) What is measured
Week 11 HW: Bioproduction and Cloudlabs
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork Make a note on your HTGAA webpages including: what you contributed to the community bioart project I change 3 pixels during the lecture, I wish I got a screen shot! what you liked about the project, and I like the real-time collaboration on something artistic