Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Question 1: 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. DNA is the rulebook of life! Imagine it as small lego pieces, building up the entire empire of a human body. But, what if we can pick out the lego pieces and assemble them in different, never-thought-before ways, to unlock new functions and outcomes ?! This is the potential of engineering synthetic genetic circuits! I am intrigued by the possibility of engineering our desired outcome by designing genetic circuits that can alter or control it.
Week 2 HW: DNA Read-Write-Edit - Pre Lecture Prep
Pre-Lecture Prep Homework Questions from Professor Jacobson: 1 Machinery of nature, for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy? The error rate for the error correcting polymerase is approximately 1 in 10^6 base pairs. The human genome is about 3 billion base pairs in lenght, meaning that without correction, a single round of replication can result in approximately 3000 errors per cell division.
Week 2 HW: DNA Read-Write-Edit - Post Lecture Learnings
Part 0: Basics of Gel Electrophoresis Attend or watch all lecture and recitation videos. Optionally watch bootcamp. Status: Completed. Part 1: Benchling & In-silico Gel Art See this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis” for details. Overview: 1 Make a free account at benchling.com 2 Import the Lambda DNA. 3 Simulate Restriction Enzyme Digestion with the following Enzymes: a EcoRI b HindIII c BamHI d KpnI e EcoRV f SacI g SalI 4 Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks. 5 You might find Ronan’s website a helpful tool for quickly iterating on designs!
Assignment: Python Script for Opentrons Artwork I used the GUI coordinates to prepare this code: Post Lab Questions: One of the great parts about having an automated robot is being able to precisely mix, deposit, and run reactions without much intervention, and design and deploy experiments remotely.
Week 4 HW: Protein Design - Part 1
Part A: Conceptual Questions 1. 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) On average, if we assume that 25% of meat is protein, then we would be taking 500*0.25 = 125 g of protein intake. With an average of 100 Da per amino acid as it’s molecular weight, the total number of moles of amino acids become 1.25 moles. Multiplying with the Avagadro’s number, we get roughly seven hundred fifty-two sextillion (7.528 * 10^23) molecules !!
Week 5: Protein Design - Part II
SOD Peptide Design from Pranam 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. Your challenge: a. Design short peptides that bind mutant SOD1. b. Then decide which ones are worth advancing toward therapy.
Week 6: Genetic Circuits - Part I: Assembly Technologies
DNA Assembly 1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Standard Phusion 2X Master Mix contains the following essential components: Phusion High-Fidelity DNA Polymerase: A specialized, proofreading enzyme coupled to a processivity-enhancing domain. Its purpose is to catalyze DNA synthesis with high speed and extremely low error rates. dNTPs (dATP, dCTP, dGTP, dTTP): The nucleotide building blocks required by the polymerase to synthesize the new complementary DNA strands. Phusion HF Buffer: An optimized reaction buffer containing MgCl2. Magnesium (Mg2+) acts as an important cofactor for the DNA polymerase enzyme, stabilizing the reaction and facilitating the smooth incorporation of dNTPs. 2. What are some factors that determine primer annealing temperature during PCR?
Week 7: Genetic Circuits - Part II: Neuromorphic Circuits
Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs outperform traditional Boolean circuits by using sophisticated, brain-like processing, with significant molecular noise reduction. Their pros include analog integration, noise filtering, pattern recognition and efficiency. By processing continuous chemical gradients compared to “on/off” signals, thus allowing cells to respond to the exact intensity of a stimulus. By integrating multiple signals, they are more robust against the random molecular fluctuations (noise) seen of the cytoplasm. IANNs can identify complex biomarker signatures, without needing a high number of logic gates. They can achieve higher computational power with fewer genetic parts, reducing the metabolic burden on the host. 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.
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. Flexibility and Control: Since there is no cell membrane, the system is an open environment, where we can directly manipulate concentrations of substrates, add non-canonical amino acids, or introduce specific inhibitors/activators with no worries about cellular transport or toxicity. Case 1: Toxic Proteins: Many proteins, such as antimicrobial peptides or certain enzymes, are lethal to host cells. CFPS allows the production of these proteins. Case 2: Rapid Prototyping: In vivo methods need time-consuming cloning, transformation, and cell culture. CFPS use’s linear DNA as a template, reducing the time from days to hours. Describe the main components of a cell-free expression system and explain the role of each component. Cell Extract (Crude Lysate): It contains ribosomes, aminoacyl-tRNA synthetases, translation factors, and tRNAs. Energy and Buffer Systems: This includes ATP and GTP , an energy regeneration substrate (like Phosphoenolpyruvate), and essential ions (Mg2+ and K+) to maintain enzymatic activity and pH. Genetic Template and Building Blocks: DNA provides the instructions, while the 20 standard amino acids provide the raw material for the protein chain. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment. Protein synthesis is energetically expensive. ATP is consumed rapidly for amino acid activation and ribosome movement, and it is also naturally degraded by other enzymes in the extract.
Week 10: Advanced Imaging and Measurement Technology
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.
Week 11: Bioproduction & Cloud Labs
The 1,536 Pixel Artwork Canvas | Collective Artwork While I don’t exactly remember what I did in the artwork, since I forgot to note it down, I remember making 2-10 edits in the artwork. It took me a little too much time to understand what was exactly happening, but I loved the whole project and the experience of doing it.
