Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Class Assignment Application: SoilBuddy With climate change set to disrupt global agriculture and the unsustainability of commercial monocultures threatening to render the arable land left unusable, humanity urgently needs solutions facilitating soil stewardship to fortify our food supply. Existing solutions are either too expensive for small-to-mid farms, worsening inequality in the developing world, impractical for massive commercial farms, necessitating guesswork set to be upended by the sea change wrought by climate change, or provide, at best, noisy, time-lagged feedback through remote sensing.
Week 2 HW: DNA Read, Write and Edit
Part 0 Done. Part 1 Since it’s passe to create “MIT” with the electrophoresis gel, I decided to reverse the order of the letters to spell “TIM” instead. Part 2 I don’t have in-person access to a node, so can’t perform the wet lab component.
Part 1 Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications. The paper I’ll be analyzing is titled “Development of a high-throughput minimum inhibitory concentration (HT-MIC) testing workflow” published in Frontiers in Microbiology. In summary, the researchers used an Opentrons OT-2 robot to automate an assay used to test the safety-efficacy profile of an antibiotic. They accomplished this by programmatically preparing a serial dilution of the antibiotic candidate across multiple 96-well plates with replicates, innoculating the wells with the test bacteria, then measuring the optical density of each well with a plate reader to determine bacterial viability after the antibiotic challenge.
Week 4 HW: Protein Design Part I
Part A Number of amino acids = 500g/(100 Da) = 5 mol = 3.0 x 10^24 molecules of amino acids (approx). Over the course of digestion, the polynucleotides are broken down into constituent nucleobases prior to absorption. Considering the degeneracy of codons, these 20 amino acids were sufficient to produce a diversity of proteins, and were evolutionarily conserved due to a lack of strong selection pressure against them (ie. they were “good enough”). . They could’ve formed abiotically given the presence of organic precursors such as carboxylic acids, amines and small molecule side groups, catalyzed by zeolites, and triggered by lightning and heat on the primordial earth or extraterrestrial asteroids respectively. Left-handed spirals. Yes, some examples are $\pi$ helices and $3_{10}$ helices. It’s more energetically stable given L-amino acids and D-saccharides predominate natural protein helices and the sugar-phosphate backbone of polynucleotides respectively. In aqueous environments, the hydrophobic portions of beta sheets are driven together by steric interactions while the hydrophilic groups facilitate relatively strong intermolecular hydrogen bonding. Beta sheets allow for strong intermolecular bonding, in hydrophobic zippers, that renders misfolded proteins more stable than their functional conformations and facilitates the addition of more proteins to the aggregate fibril - the biochemical basis of amyloid pathogenesis. Yes, you could make materials out of amyloid beta sheets but they’d only be stable under aqueous conditions and would be potential biotoxins. . Part B I chose to investigate an antifreeze protein from an arctic bacterium as a potential solution for winter snow-clearing.
Week 5: HW Protein Design Part II
Part A Part 1: Generate Binders with PepMLM Binder Sequence Perplexity Score WRYYATVARHKE 15.38014901 WLYYVVVLRHGE 32.45105037 WRYYAAGARLKE 11.75101427 WRYYATAVELKG 10.44080960 Part 2: Evaluate Binders with AlphaFold3 Peptide ID Binder Sequence ipTM Score Location Peptide 0 WRYYATVARHKE 0.35 Approaches the $\beta$ barrel; surface-bound Peptide 1 WLYYVVVLRHGE 0.33 Approaches the $\beta$ barrel; surface-bound Peptide 2 WRYYAAGARLKE 0.35 Approaches the $\beta$ barrel; surface-bound Peptide 3 WRYYATAVELKG 0.22 Conforms to the dimer interface; binds to a pocket Control FLYRWLPSRRGG 0.31 Surrounding the $\beta$ barrel; surface-bound While peptides 0-2 exceed the control (known binder)’s ipTM scores, indicating stronger protein-protein interaction, they still represent low confidence values. I would not conclude that they produce stronger protein-protein interactions for use as potential molecular glues.
Week 6 HW: Genetic Circuits Part I
Assignment: DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The NEB 2X master mix includes engineered thermostable DNApol to synthesize complimentary strands during the extension phase of PCR, with low error rate and high expressivity to enable faster, more accurate DNA replication. It includes a buffer to maintain optimum pH for enzymatic catalysis and prevent the denaturation of enzyme or nucleic acids, as well as ${Mg}{Cl}_{2}$ to provide $Mg^{2+}$ as a cofactor for DNApol.
Week 7 HW: Genetic Circuits Part II
Intracellular Artificial Neural Networks (IANNs) 1. What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs more easily provides for graded responses and can scale intelligent responses; they can approximate every mathematical function and not just step functions.
Homework Part A Assignment 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. It lacks the stress response, proteostasis and detoxification of an in vivo environment, allowing for much higher expression of protein including toxic varieties or those incorporating xenobiotic compounds, as in bio-orthogonal experiments. Reaction conditions are standard and can be externally controlled/varied (eg temperature) more precisely than in in vivo systems. The progress of reaction is more easily followed using imaging techniques, allowing for rapid optimization and sequential syntheses. Two cases where cell-free expression is better than cell production are the expression of protein toxic to bacterial/fungal chassis (eg antibacterial/antifungal compounds) as well as proteins intended for use in human medicine (free of endotoxins).
Week 10 HW: Imaging and Measurement
Waters Lab Part I 28006.60Da Waters Lab Part II Waters Lab Part III Waters Lab Part IV Waters Lab Part V