Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
- First, describe a biological engineering application or tool you want to develop and why. This could be inspired by an idea for your HTGAA class project and/or something for which you are already doing in your research, or something you are just curious about. Inspired by the MELiSSA project (Micro-Ecological Life Support System Alternative) from ESA, this project proposes an ecosystem composed of microorganisms and higher plants using their metabolic waste products as a substrate for the next compartment. This project is designed to study the behavior of artificial ecosystems and to develop the technologies required for future regenerative life-support systems in long-duration human space missions, such as lunar bases or missions to Mars. The system comprises five different compartments, each one colonized respectively by anoxygenic thermophilic bacteria, photoheterotrophic bacteria, nitrifying bacteria, photosynthetic bacteria, higher plants, and the human crew. I would like to conceptually integrate these microorganisms and higher plants with a plasmids-based control system, through the use of reporter genes and inducible regulatory elements. This would increase the security (allowing real-time monitoring of metabolics states, for example) and predictability of the system.
Week 2 HW: DNA read, write and edit.
Week 02 - Lecture Questions Professor Jacobson The fidelity of DNA replication is governed by DNA polymerase and its associated repair systems. The intrinsic error rate of DNA polymerase, in the absence of proofreading, is approximately 10-4 to 10-5 per nucleotide. In eukaryotes, replicative polymerases utilize 3’ —} 5’ exonuclease activity for proofreading, which enhances fidelity to an error rate of approximately 10-7. When integrated with post-replicative mismatch repair (MMR) mechanisms, the effective error rate is further optimized to roughly 10-9 to 10-10 per nucleotide.Given that the human genome comprises approximately 3.2 x 109 base pairs, replication without these multi-layered fidelity mechanisms would result in a mutational load incompatible with cellular viability. Biological systems mitigate this risk through a hierarchy of safeguards—polymerase proofreading, mismatch repair, and various DNA damage response pathways—ensuring that the mutation rate per genome remains within a range that sustains evolutionary stability and life. A typical human protein consists of approximately 300 to 400 amino acids. Due to the degeneracy of the genetic code—where 64 codons encode 20 amino acids—the theoretical number of DNA sequences capable of encoding a single protein is exceptionally high. However, functional constraints significantly restrict this theoretical diversity. Key limiting factors include:
Week 03 - Python Script for Opentrons Artwork I was not able to write the code entirely by myself. The closest I got was generating concentric circles, wich reminded me of the Argentine “Escarapela” (with the help AI). My original idea, however, was to made an Argentine Mate which I did in https://opentrons-art.rcdonovan.com/ I also did a Cherry!
Week 4 HW: Protein Design - Part I
Week 04 - 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) 500 g of meat has more or less 22% of protein, so 500 g x 0.22 =110 g of protein Average amino acid ≈ 100 Daltons and 1 Dalton ≈ 1 g/mol, so 100 Da≈100 g/mol, in order to convert grams of protein to moles of amino acids
Week 5 HW: Protein Design Part II
Week 5 Part A: SOD1 Binder Peptide Design (From Pranam) Part 1: Generate Binders with PepMLM Begin by retrieving the human SOD1 sequence from UniProt (P00441) and introducing the A4V mutation. Using the PepMLM Colab linked from the HuggingFace PepMLM-650M model card: Generate four peptides of length 12 amino acids conditioned on the mutant SOD1 sequence. To your generated list, add the known SOD1-binding peptide FLYRWLPSRRGG for comparison. Record the perplexity scores that indicate PepMLM’s confidence in the binders.
Week 6 HW: Genetic Circuits: Part I
Week 6 — Genetic Circuits Part I: Assembly Technologies DNA Assembly Answer these questions about the protocol in this week’s lab: What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The Phusion High-Fidelity PCR Master Mix contains several components: Phusion DNA polymerase → a high-fidelity enzyme that synthesizes DNA with very low error rates (With a failure rate 50 times lower than Taq and 6 times lower than Pfu, these polymerases are an excellent choice for cloning and other applications requiring high fidelity), which is critical when amplifying fragments of the amilCP gene. dNTPs (deoxynucleotide triphosphates) → building blocks for new DNA strands MgCl₂ → cofactor necessary for polymerase activity Buffer system → maintains optimal pH and ionic conditions These components work together to ensure accurate and efficient DNA amplification, also Phusion DNA polymerases offer robust performance with short protocol times, even in the presence of PCR inhibitors. They generate higher yields with less enzyme than other DNA polymerases. In this protocol, the master mix is used to amplify amilCP fragments that will later be assembled using Gibson Assembly. What are some factors that determine primer annealing temperature during PCR? Primer annealing temperature depends on: Primer length → longer primers have higher melting temperatures, GC content → higher GC increases stability and raises Tm. Higher melting temperatures are caused due to stronger hydrogen bonding. In this protocol, primers include additional overhangs (20–22 bp) for Gibson Assembly, but only the binding region determines the annealing temperature. The annealing temperature is typically set a few degrees below the melting temperature (Tm) to ensure specific binding. 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. In this protocol, PCR amplify specific regions of the amilCP gene, including mutated regions in the chromophore, allowing precise control over sequence design In contrast, restriction digestion (using PvuII) is used to linearize the pUC19 plasmid backbone. PCR is more flexible and allows introduction of mutations and overlaps, while restriction digestion relies on specific enzyme recognition sites. PCR is preferable for designing new constructs, whereas digestion is useful for preparing existing plasmid backbones.
Week 7 HW: Genetic Circuits: Part II
Assignment Part 1: Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? Traditional genetic circuits primarily rely on Boolean logic (AND, OR, NOT gates), which results in “all-or-nothing” digital responses. Intracellular Artificial Neural Networks (IANNs) offer several distinct advantages: Non-linear Signal Integration: Unlike Boolean gates that require strict thresholds, IANNs use activation functions (like Hill functions) to process analog chemical gradients, allowing for more nuanced environmental sensing. Weighted Inputs: IANNs allow for “tunable” inputs. By varying promoter strength or ribosome binding site (RBS) efficiency, the cell can assign different weights (w) to various biological signals, prioritizing one metabolite over another. Noise Filtering: Biological environments are inherently “noisy.” The summation and thresholding architecture of a perceptron acts as a natural buffer, preventing the circuit from misfiring due to minor stochastic fluctuations in gene expression. Computational Density: A single-layer IANN can perform complex classifications that would require a much larger and more metabolically taxing combination of traditional logic gates. 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: An engineered E. coli strain that acts as a therapeutic diagnostic tool within the human gut.
Homework Part A: General and Lecturer-Specific Questions 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. The primary advantage lies in the decoupling of the reaction from cellular metabolism. Flexibility: It allows the use of linear DNA, eliminates the need for transformation and host-specific codon optimization, and facilitates the expression of proteins that are toxic to the host. Control of variables: It is an “open” system. You can manipulate buffer composition (pH, ionic strength), add chaperones, modify the Mg2+/K+ ratio, or add specific redox agents for disulfide bond formation in real-time, without the limitations of cellular homeostasis. Use cases: Toxic proteins: Production of proteins that compromise host viability (e.g., antimicrobial peptides or nucleases). Non-canonical amino acid (ncAA) incorporation: Facilitates genetic code expansion via stop codon suppression without competition from endogenous tRNAs.
Week 10 HW: Advanced Imaging & Measurement Technology
Week 10 — Advanced Imaging & Measurement Technology Homework: Final Project 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. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail. Measurement and Validation Techniques for the Bio-Sticker Controlled Gas Exposure Assays The Bio-Sticker will first be tested in sealed exposure chambers containing precisely known concentrations of target toxic gases, such as ammonia or formaldehyde. These chambers allow accurate simulation of hazardous industrial environments while maintaining strict control over temperature, humidity, and gas concentration. By exposing the engineered fungal Bio-Sticker to increasing concentrations of the target analyte, we can determine its activation threshold, sensitivity, and dynamic range. This approach also enables the generation of dose-response curves, which are essential for calibrating the system and defining the concentration at which the color change becomes visible. Colorimetric Analysis The primary readout of the Bio-Sticker is the visible blue color produced by expression of the chromoprotein AmilCP. Colorimetric analysis will be used to quantify this response objectively. Images of the Bio-Sticker will be captured under standardized lighting conditions, and software such as ImageJ will be used to analyze changes in color intensity. Measurements will focus on RGB (red, green, blue) values and, when applicable, absorbance at the wavelength corresponding to AmilCP. This technique allows precise quantification of signal strength, comparison between samples, and monitoring of signal development over time. Digital Image Analysis In addition to simple colorimetric measurements, digital image processing will be employed to evaluate spatial uniformity, signal progression, and long-term stability of the color response. Time-course imaging can be used to track the kinetics of AmilCP expression after exposure to toxic gases. This enables measurement of response time, persistence of the signal, and any degradation or fading over extended periods. Such analyses are particularly important for assessing practical usability in field conditions. Polymerase Chain Reaction (PCR) PCR will be used to confirm successful integration of the engineered genetic circuit into the Aspergillus nidulans genome. Specific primers will be designed to amplify regions spanning the inserted construct and adjacent genomic sequences. Successful amplification of fragments of the expected size will verify the presence of the biosensing cassette. This serves as an initial molecular confirmation that the strain has been correctly engineered. DNA Sequencing Following PCR confirmation, DNA sequencing will be performed to verify the exact nucleotide sequence of the inserted construct. This step ensures that the promoter, sensing elements, reporter gene (AmilCP), and regulatory sequences have been integrated without mutations, deletions, or rearrangements. Sequence verification is critical to ensure that the genetic circuit will function as intended. Reverse Transcription Quantitative PCR (RT-qPCR) RT-qPCR will be used to measure transcriptional activation of the reporter gene after gas exposure. RNA will be extracted from the fungal cells before and after exposure to target gases, converted into complementary DNA (cDNA), and amplified using gene-specific primers. By comparing transcript levels under different conditions, this technique will quantify the extent to which the sensing circuit is activated. RT-qPCR provides highly sensitive, quantitative insight into gene expression dynamics. Spectrophotometry (Optional) Spectrophotometric analysis may be used to complement image-based measurements. Pigments extracted from fungal samples can be analyzed by measuring absorbance at wavelengths specific to AmilCP. This provides an additional quantitative assessment of chromoprotein production and can be particularly useful for validating colorimetric data. Specificity Testing To ensure selectivity, the Bio-Sticker will be exposed not only to target toxic gases but also to non-target compounds commonly present in industrial environments. By comparing responses across these conditions, we can determine whether the system selectively responds to the intended analyte or produces false positives. This is essential for establishing reliability in real-world applications. Stability and Shelf-Life Testing Long-term performance will be evaluated by monitoring the Bio-Sticker under different storage and environmental conditions. Parameters such as baseline color, response capability, and signal durability will be assessed over time. These studies will determine shelf life, operational stability, and robustness under field deployment conditions. Together, these techniques will provide a comprehensive characterization of the Bio-Sticker, from genetic validation to functional performance, ensuring that it operates as a reliable, low-cost, and easily interpretable biosensor for toxic gas detection in hazardous industrial environments. Homework: Waters Part I — Molecular Weight We will analyze an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).
Week 11 HW: Bioproduction & Cloud Labs
Week 11 — Bioproduction & Cloud Labs Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork Unfortunately, I couldn’t contribute but I think it’s a great project that improves creativity and working in teams. The best part of it is there’s a contribution from all over the world. I think for next year we could have a more detailed explanation of the draw-to-made in order to create something specific but with different points of view. For example to create a plate to draw a bacteria and see what happens. I think this would be interesting.