Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Governance Assignment Biological Engineering Application Immunotherapies are a promising avenue in cancer treatment as they leverage the immune system’s innate ability to recognize and target non-self structures. However, traditional immunotherapies often result in on-target off-tumor effects, particularly in solid tumors. Synthetic biology has enabled new avenues of discovery to minimize this immunotherapy-related toxicity: engineering immune cells to target tumor-associated antigens (TAAs) or engineering genetic circuits to detect cancer disease signatures (Zhu et al., 2024). For example, modifying the traditional Chimeric Antigen Receptor (CAR) T-cell immunotherapy approach with a synthetic Notch (synNotch) receptor has demonstrated the ability to suppress off-target cytotoxicity related to organ rejection (Reddy et al., 2024) and selectively target cancerous cells in the central nervous system of mice rather than elsewhere in the body (Simic et al., 2024). Yet, while synNotch-modified CAR-T therapies show promise in their ability to reduce immunotherapy-related toxicity, additional research is needed to effectively administer these bioengineered cell systems in patients beyond pre-clinical experimentation.
Week 2 HW: DNA Read, Write, and Edit
Part 1: Benchling and In-silico Gel Art Virtual restriction enzyme digest designed with DNA from the bacteriophage Kampy (isolated at W&M!) and the restriction enzymes BstXI, KpnI, and SfiI to resemble two bacteriophages. The chosen restriction enzymes were selected because they were in stock at William & Mary, had multiple cut sites in the Kampy DNA, and could be combined to make a design resembling a bacteriophage.
Python Script for Opentrons Artwork My Opentrons design is meant to resemble a frog because I use Xenopus laevis as my model organism in my honors thesis research at William & Mary. Post-Lab Questions Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications. In Sanders et al., 2022, the researchers use an Opentron robot to optimize a bacterial whole-genome sequencing (WGS) protocol for gut microbiota samples. The Opentron was used for DNA extraction and library preparation steps, reducing the overall cost of WGS by ~$10 per genome and eliminating the need for 16S rRNA gene-based screening.
Week 4 HW: Protein Design Part I
Part A: Conceptual Questions Part B: Protein Analysis and Visualization Part C: Using ML-Based Protein Design Tools C1: Protein Language Modeling C2: Protein Folding C3: Protein Generation Part D: Group Brainstorm on Bacteriophage Engineering I collaborated with Heather Qian on this assignment!
Week 5 HW: Protein Design Part II
Part A: SOD1 Binder Peptide Design Part 1: Generate Binders with PepMLM SOD1_A4V Sequence: MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Peptide Perplexity Score WHYYVAVVRLGE 36.758428 WLYPPTAVAHKK 14.060910 WRYYPVALAHKK 11.940822 HRYPAVVVEHKE 16.230484 FLYRWLPSRRGG 20.635231 Part 2: Evaluate Binders with AlphaFold3 Binder ipTM Score Binding Site Evaluation 1 0.71 Binder 1 associates near the β barrel at the surface of the protein. 2 0.44 Like Binder 1, Binder 2 associates near the β barrel at the surface of the protein. However, this binder has a much lower ipTM score, meaning the confidence in this generated structure is much lower. 3 0.31 Binder 3 associates across the β barrel and disordered region at the surface of the SOD1 protein. 4 0.30 Binder 4 associates at the surface of the disordered region. 5 0.39 Binder 5 also associates at the surface of the disordered region. Binders 1 and 2 have stronger ipTM values than Binder 5, the known binder provided for this exercise. All binders associate near the surface of the SOD1 protein, not integrating into the protein interior. The ipTM value for Binder 1 is relatively strong (0.71), meaning there is high confidence in that association between SOD1 and the binder.
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: uses dNTP monomers to synthesize new DNA strands dNTPs: monomers of new DNA strand Buffer: stabilizes the pH of the reaction for optimal enzymatic function MgCl2: Cofactor for the polymerase; optimizes enzymatic function and primer annealing What are some factors that determine primer annealing temperature during PCR? GC content (higher GC content = more hydrogen bonds = stronger primer annealing = higher annealing temperature) Primer length (longer primers = more hydrogen bonds = stronger primer annealing = higher annealing temperature) 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. PCR: Enzyme: DNA polymerase DNA polymerases bind to primers and synthesize complementary DNA to the template strand Purpose: amplify! Polymerases synthesize DNA by recognizing primers (designed to flank the specific region of interest) and incorporating dNTPs into a novel DNA strand When to use: To detect a specific sequence within a mixed sample To create more of a specific DNA sequence (amplify) Restriction enzyme: Enzyme: restriction endonuclease Restriction endonucleases recognize specific nucleotide sequences (4-8 bp) in double-stranded DNA and cut in a specific pattern (blunt or sticky ends, depending on the enzyme) Purpose: cut! Restriction enzymes cut existing template DNA, they do NOT amplify DNA fragments When to use: To linearize bacterial plasmids (ex. for in vitro transcription of capped mRNA for microinjection into X. laevis!) To cut DNA fragments to assemble/ligate together and transform into a plasmid How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Primer design! The 5’ tail overhang of each primer should be identical to the adjacent DNA fragment the 3’ end of the primer should be complementary to the DNA fragment to which it will anneal Restriction Digest: Restriction enzyme cut sites should be avoided PCR: Primers should be designed that flank the overhang regions How does the plasmid DNA enter the E. coli cells during transformation? Heat shock transformation - briefly raising temperature increases the permeability of the bacterial cell wall, allowing the plasmid DNA to enter into the E. coli cell E. coli cell sample placed on ice for ~15-30 minutes Sample heated to 42ºC for ~15-30 seconds Sample then returned to ice for ~5 minutes Describe Golden Gate Assembly Golden Gate Assembly utilizes Type IIS restriction enzymes (such as BsaI) which recognize non-pallindromic sequences, cut outside the recognition site (to avoid damaging the DNA sequence of interest), and create sticky ends of variable lengths. First, template DNA is amplified using PCR with primers specified to include the TIIS recognition and cut sites. Next, a restriction digest with the TIIS enzyme is performed to create DNA fragments with sticky ends. The sticky ends of adjacent fragments should be complementary so the sequences can be ligated in the appropriate order. Following ligation of DNA fragments with a plasmid, the engineered plasmid can be transformed into a bacterial cell (ex. E. coli), and bacterial colonies containing the plasmid can be screened for furhter use. Image generated by ChatGPT
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? Variables input into Boolean functions are binary (either ON or OFF), whereas the variables input into IANNs can be continuous. Additionally, IANNs can input multiple variables at a time while Boolean functions (like a two-layer IANN) only integrate two variables.