Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Important: I use ChatGPT and Gemini to help me organize my ideas, translate some concepts and reunite everything!!
Week 2 HW: DNA Read, Write and Edit
–> This image shows DNA fragments separated by agarose gel electrophoresis and stained with a fluorescent dye, performed during my Genetic Engineering course. Part 1: Benchling and In-silico Gel Art According to the instructions, this is the Gel I designed for p53 human protein (tried my best to make it look like a butterfly i’m sorry!!) Part 3: DNA Design Challenge
Week 3: Lab Automation Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications. Olsen J.V. et al. Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics (2024) Mol Cell Proteomics 23(7), 100790. DOI 10.1016/j.mcpro.2024.100790 This article describes a fully automated workflow for preparing clinical proteomics samples, from protein digestion to loading peptides into Evotips (disposable, tip-based C18 reversed-phase trap columns), ready for LC-MS/MS analysis. They use the Opentrons OT-2 liquid-handling robot, which controls all preparation steps without manual intervention after the initial loading of reagents. The process combines protein capture through aggregation on magnetic beads with enzymatic digestion and, without centrifugation steps, directly transfers the peptides to Evotips using positive pressure, all programmed through downloadable scripts from the Evosep website. Using this method, up to 192 samples can be processed in parallel in approximately 6 h, which equals to 100 samples/day and eliminates human variability. In tests with HeLa lysates, the workflow identified ~8.000 protein groups and ~130.000 peptides using an 11.5-min gradient on the Orbitrap Astral, demonstrating high sensitivity and reproducibility. It was also applied to 192 plasma samples from patients with metastatic melanoma, revealing clinically relevant protein changes.
Week 4 HW: Protein Design Part I
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) Meat has an average 20% of protein, so 500 grams of meat would have 100 grams of protein. An average amino acid mass is 100 Da (100g/mol). So, according to Avogadro’s number, in a 500 g piece of meat, we are consuming approximately 6.62x1023 molecules of amino acids. Why do humans eat beef but do not become a cow, eat fish but do not become fish? Dietary proteins are digested into individual amino acids and absorbed and reused to build proteins used for the human metabolism, processes that determine our identity, not the food we eat. Why are there only 20 natural amino acids? There are 20 natural amino acids (22 in some organisms) because the standard genetic code evolved to use this 20 structures, maybe because they were efficient for metabolism, providing sufficient chemical diversity or they were optimal in terms of evolution. Can you make other non-natural amino acids? Design some new amino acids. Using synbio it is possible to create new amino acids according to what you need or want to do, for example maybe metal-binding amino acids to coordinate metal ions, or photo-crosslinking amino acids to form bonds when exposed to light, or perhaps adding electronegative elements like Fluor to alter their electronic properties. If you make an α-helix using D-amino acids, what handedness (right or left) would you expect? L-amino acids form right-handed helices, while D-amino acids would form a left-handed helix. Can you discover additional helices in proteins? Synbio help us explore and find new structural possibilities since protein folding is certainly a complex issue that, at present, is not fully understood. Even though they are rare, it is possible to find -helix, 310 helix, foldamers (artificial oligomers), and maybe there are more out there to be discovered. Why are most molecular helices right-handed? Because biological amino acids are L-chiral, making right-handed helices more sterically favorable and stable energetically. Why do β-sheets tend to aggregate? What is the driving force for β-sheet aggregation? β-sheets are made of protein strands that lie next to each other and are connected by many hydrogen bonds, forming a very flat and extended surface with many places where it can stick to another sheet, so they can easily attach and stack together. Some amino acids in proteins are hydrophobic and when the β- sheets form, these parts can be exposed. To avoid water, they stick to each other hiding from water. Aggregation happens because it is energetically favorable, so the driving forces are the hydrophobic effect, the hydrogen bonds to increase stabilization and sticking together to lower the free energy. Why do many amyloid diseases form β-sheets? Can you use amyloid β-sheets as materials? In amyloid diseases (like Alzheimer’s) some proteins misfold, losing their normal shape and refolding into β-sheet structures. Since these structures are very stable, it is hard to go back from there. These misfolded proteins then stick to each other, form long fibers (amyloid fibrils), accumulate in tissues and damage cells. Since these structures are very strong, stable and able to self-assemble, it is possible to use them as materials. Scientist are studying them for creating nanofibers, biomaterials, tissue scaffolds, and drug delivery systems. Part B. Protein Analysis and Visualization
Week 5 HW: Protein Design: Part II
PART A: SOD1 Binder Peptide Design Superoxide dismutase 1 (SOD1) is a cytosolic antioxidant enzyme that converts superoxide radicals into hydrogen peroxide and oxygen. In its native state, it forms a stable homodimer and binds copper and zinc. Mutations in SOD1 cause familial Amyotrophic Lateral Sclerosis (ALS). Among them, the A4V mutation (Alanine → Valine at residue 4) leads to one of the most aggressive forms of the disease. The mutation subtly destabilizes the N-terminus, perturbs folding energetics, and promotes toxic aggregation.
Week 6 HW: Genetic Circuits: Part I
What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? This master mix contains a Phusion high-fidelity DNA polymerase, an enzyme that synthesizes new DNA strands during PCR and has proofreading activity that reduces the error rate compared to standard polymerases. The enzyme needs Mg+2 ions as a cofactor required for the polymerase activity. This mix also contains dNTPs (deoxynucleotide triphosphates), which are the building blocks used by the polymerase to synthesize new DNA. Another key component is the reaction buffer, which maintains the optimal pH and salt conditions required for the enzyme to function properly. Finally, water is used as the solvent. What are some factors that determine primer annealing temperature during PCR? The annealing temperature mainly depends on the melting temperature (Tm) of the primers, which is influenced by: Primer length: longer primers have higher melting temperatures G-C content: G-C base pairs form three hydrogen bonds increasing primer stability (but more energy is required to hydrolyze them) compared to A-T pairs. Sequence composition and presence of secondary structures (like hairpins or dimers) Salt concentration and reaction conditions Usually the annealing temperature is chosen to be a few degrees lower than the calculated Tm of the primers to allow efficient binding while maintaining specificity. 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 and restriction enzyme digestion are both methods to generate linear DNA fragments, but working differently. PCR amplifies a specific DNA region using primers and a DNA polymerase. The primers determine the exact boundaries of the fragment being amplified, which makes PCR very flexible. This technique is useful when we want to amplify a specific gene or add sequences such as overlaps or tags to the ends of the DNA. Restriction enzyme digestion uses these enzymes that cut DNA at specific recognition sequences. This method requires that the restriction sites already exist in the sequence. The protocol usually involves incubating the DNA with the enzyme under optimal buffer and temperature conditions. In terms of when to use each method, PCR is preferable when we need custom DNA fragments, sequence modification, or large amplification of DNA. Restriction digestion is often sued when we want to cut plasmids or DNA molecular at defined natural restriction sites, especially when preparing vectors for cloning. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? To ensure this fragments are compatible, they must contain overlapping homologous regions at their ends. Typically, these overlaps are about 20-40 base pairs long and are designed to match the adjacent DNA fragment. It is possible to design these PCR primers that include the necessary overlap sequences at their 5’ ends. After amplification, the primers will contain the overlaps needed for assembly. It is also important to check that the fragments are correctly sized and free of unwanted sequences using gel electrophoresis. How does the plasmid DNA enter the E. coli cells during transformation? During transformation plasmid DNA enters E. coli cells when the bacterial membrane becomes temporarily permeable. In chemical transformation, cells are first treated with calcium chloride, which helps neutralize the negative charges of both the DNA and the cell membrane. Then a heat shock step is applied, that creates a sudden temperature change that helps DNA molecules pass through the membrane and enter the cell. There are other methods such electroporation, a short electrical pulse applied to the cells in order to create temporary pores in the membrane, allowing plasmid DNA to enter the cytoplasm. Describe another assembly method in detail (such as Golden Gate Assembly) a. Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
Week 7 HW: Genetic Circuits: Part II
Part 1: Intracellular Artificial Neural Networks (IANNs): What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? These IANNs have several advantages compared to traditional genetic circuits: Continuous responses instead of binary outputs, which makes them more similar to real biological systems, where gene expression is not just “on or off” but varies in intensity. Better handling of noisy biological environments, because IANNs can integrate multiple inputs and average signals, making them more robust to fluctuations caused by these noisy systems. Ability to learn complex patterns compared to Boolean circuits that are limited to simple logic, while IANNs can approximate complex nonlinear functions allowing more sophisticated decision-making. This neural-like architectures can be extended to multiple layers, enabling hierarchical processing. IANNs are more biologically realistic, since gene regulatory networks in cells already behave more like analog systems. 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. Application: Smart cancer cell detection and response system
GENERAL HW 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. CFPS transitioned from a “black box” scenario to an open controllable system, so flexibility and control are the main advantages here. This allows us to precisely manipulate the concentrations of amino acids, salts, and templates. It also allows for the addition of non-canonical amino acids or cytotoxic agents that would otherwise kill a living host. Cases where CFPS is more beneficial:
Week 10 HW: Advanced Imaging and Measurement Technology
Homework: FINAL PROJECT One of the main aspects to measure in this work is the expression level of the APP since the goal of the CRISPRi system is to reduce its transcription. This could be measured using qPCR (mRNA levels of APP), extracting the RNA from treated and control cells, performing reverse transcription and quantifying gene expression differences. It is also important to measure whether changes in gene expression translate into protein level changes, using Western Blot to detect APP protein levels or ELISA to quantify amyloid-beta peptides.
Week 11 HW: Bioproduction & Cloudlabs
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork In the initial artwork (before the complete design was changed :()) I made that little flower using mRFP, sfGFP and mKO2. I actually loved this project because we had the opportunity to contribute to the HTGAA effort. I would also like to say that Ronan’s page is easy to understand and use, thanks Ronan!