Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Governance 1. Bioengineering Tool Phage satellites are a diverse class of mobile genetic elements that parasitize a phage. Extracellular Prophage-Inducing Particles (EPIPs), are a novel class of phage satellite discovered by the Saha Lab that induces the extremely stable prophage, HerbertWM, in Mycolicibacterium aichiense. Due to their novelty, much of their mechanisms of action are unknown, but it is hypothesized that they contain antirepressors or partial antirepressors due to annotated genes that bear resemblance to BRO domains which have been noted to have some influence over transcription (Zemskov et al., 2000). Because of the observed ability of EPIPs to induce a stable prophage, developing a lytic-lysogenic switch from their putative antirepressors would be valuable for many applications including medical and environmental applications. This switch could ensure that a temperate phage lyses bacterial pathogens in phage therapy or control the transcription of engineered constructs in the soil.
Week 10 HW: Imaging and Measurement
For properly formatted equations, read the version on GitHub. 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 would measure both fluorescence and OD600. Fluorescence will tell me how well the antirepressors are being expressed, and OD600 will tell me how much the antirepressors affecting bacterial growth.
Week 2 HW: DNA Read, Write, and Edit
DNA Design Challenge Protein chosen: tape measure protein of Alyssa1, a Mycolicibacterium phage satellite called an “Extracellular Prophage-Inducing Particle” (EPIP). Tape measure proteins are rare in phage satellites, and TEM imaging has shown that EPIPs have uniquely long tails compared to the helper phage, the phage they parasitize, suggesting the tape measure protein may contribute to a unique mechanism of parasitism.
Paper Discussion Paper: AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots This paper reports using the Opentrons robot to conduct PCR, Golden Gate assembly, and Gibson assembly. It appears that the robot was able to determine specific parameters for experiments such as annealing temperature. The paper did not go into much detail about how this occurred, but after looking at some of the supplementary materials, the script likely parses .csv files that include primer and fragement sequences.
Week 4 HW: Protein Design Part I
Conceptual Questions Why do humans eat beef but do not become a cow, eat fish but do not become fish? When humans eat, the macromolecules the beef are made of are broken down during digestion into monomers. These monomers are common to all life, and humans use them to build human-specific macromolecules. Why are there only 20 natural amino acids? These 20 amino acids are what evolution happened to select for. These 20 amino acids happen to be enough to build all the proteins that are necessary for life that has evolved on Earth. Theoretically, there could be more, but in our “system” of life, these 20 are enough.
Week 5 HW: Protein Design Part II
SOD1 Binder Peptide Design (From Pranam) Generate Binders with PepMLM PepMLM Colab File SOD1 sequence: MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Sequence with A4V mutation: MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Binders, perplexity score: WRVPPAALRHKE, 22.653588 HRSPPVAAEHWK, 19.512332 WRYYPVAAAWKK, 11.081843 WRYYVAALRHGK, 15.691672 known SOD1 binder: FLYRWLPSRRGG, 20.635231 Evaluate Binders with AlphaFold3 Binder ipTM Score Binding Location WRVPPAALRHKE 0.39 near β-barrel and globular part HRSPPVAAEHWK 0.26 near β-barrel, across the seam and onto globular part WRYYPVAAAWKK 0.28 near β-barrel, across the seam WRYYVAALRHGK 0.30 near β-barrel, across the seam known: FLYRWLPSRRGG 20.635231 0.31 The ipTM score is highest for the first binder, but none of the binders bind in a similar location compared to the known binder. All of the predicted binders bound a similar part of the protein that was very different from where the known binder is predicted to bind.
Week 6 HW: Genetic Circuits Part I
DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? DNA polymerase - amplifies DNA by adding nucleotides dNTPs - bases added by DNA polymerase Buffer - maintains pH and contains cofactors the DNA polymerase requires What are some factors that determine primer annealing temperature during PCR? Primer length and GC content.
Week 7 HW: Genetic Circuits Part II
Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs are able to process multiple inputs and gradients, unlike Boolean functions that largely process “black or white” options. This allows them to make more nuanced “decisions.”" 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. In cell-free protein synthesis, variables such as pH, ion concentration, components, etc. are more adjustable. Additionally, researchers can add or remove components at any time. Two cases where cell-free expression is more beneficial than cell production are when the protein is toxic or when large amounts of protein are needed quickly.