Homework
Weekly homework submissions:
Week 01 HW: Principles and Practices
Class Assignment 1. First, describe a biological engineering application or tool you want to develop and why. Endometriosis is an inflammatory disease characterized by the endometrial-like tissue growth outside of the uterine cavity. This ectopic growth leads to hormonal imbalances, systemic inflammation, and debilitating pain during menstruation, sexual intercourse, and bodily functions . Although it affects 10–15% of reproductive age women, there is currently no cure and the diagnosis of this diseases remains a clinical challenge [1]. Current clinical management is limited to hormonal suppression, pain control and surgical excision [2]. Consequently, there is a critical need for non-invasive, targeted therapies that can modulate the immune response and minimize recurrence rates without compromising the patient’s reproductive health.
Week 02 HW: DNA READ, WRITE AND EDIT
Part 1: Benchling & In-silico Gel Art This DNA gel art was designed in the style of Paul Vanouse’s Latent Figure Protocol. I chose to create the letter “P” as it is the initial of my name, Paula. To achieve this, I used Ronan’s website, which was a helpful tool for quickly iterating on the designs and determining the best enzyme combinations to form the silhouette of the letter.
Your task this week is to Create a Python file to run on an Opentrons liquid handling robot. 1. Generate an artistic design using the GUI at opentrons-art.rcdonovan.com. 2. Using the coordinates from the GUI, follow the instructions in the HTGAA26 Opentrons Colab to write your own Python script which draws your design using the Opentrons. Link to cell: https://colab.research.google.com/drive/1-f-vpwBCOx1gmlD5qXbW5z-7sLun1xwP?authuser=2#scrollTo=pczDLwsq64mk&line=1&uniqifier=1
Week 04 HW: Protein Design part I
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) The average composition of muscle without external fat cover is composed of approximately 70% water, 20% protein, and 9% fat (The exact values vary depending on the animal source) [1]. Therefore 500 g of meat provides 100 g of protein. Since proteins are chains of amino acids, once digested they break down into individual amino acid molecules. We are told the average molecular weight of an amino acid is ~100 Daltons, which means its molar mass is 100 g/mol.
Week 05 HW: Protein Design part II
Part A: SOD1 Binder 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.
Week 07 HW: Genetic circuits part II
Assignment Part 1: Intracellular Artificial Neural Networks (IANNs) 1. What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? Boolean functions are limited to discrete on/off states while IANNs are capable of processing analogue signals and, because of that, carry more information. Real world phenomena are analog, inside a cell there is inherent molecular noise, and Boolean circuits are fragile to this, especially at low signal concentrations.
Week 10 HW: Imaging and Measurement
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. —————————————————————————————————————————————————- - Toehold switch activation: Does the switch open specifically in response to LncRNA H19? I would order the toehold switch construct from Twist and it will be expressed at Ginkgo using a PURExpress cell-free reaction with and without the H19 trigger, using sfGFP as reporter. Technology: fluorescence spectroscopy with a plate reader.
Part C: Planning the Global Experiment | Cell-Free Master Mix Design 1. Given the 6 fluorescent proteins we used for our collaborative painting, identify and explain at least one biophysical or functional property of each protein that affects expression or readout in cell-free systems. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each) 1. sfGFP 2. mRFP1 3. mKO2 4. mTurquoise2 5. mScarlet_I 6. Electra2 Fluorescent protein Maturation (min) pKa Brightness Description sfGFP 13.6 54.15 Rapidly-maturing weak dimer mRFP1 60.0 4.5 12.5 Slowly-maturing monomer with low acid sensitivity mKO2 108.0 5.5 39.56 Moderate acid sensitivity mTurquoise2 33.5 3.1 27.9 Rapidly-maturing monomer with very low acid sensitivity mScarlet_I 174.0 5.3 70 Moderate acid sensitivity Electra2 61.48 3. Create a hypothesis for how adjusting one or more reagents in the cell-free mastermix could improve a specific biophysical or functional property you identified above, in order to maximize fluorescence over a 36-hour incubation. Clearly state the protein, the reagent(s), and the expected effect. I choose mKO2 because it is acid sensitive and has a slow maturation time.. My hypothesis is that increasing the HEPES-KOH concentration in the 36-hour master mix would help maintain the pH closer to 7.5 throughout the reaction. Which is important because in cell free reactions pH decreases over long incubations due to the accumulation of acidic metabolic byproducts and mKO2 slow maturation time means most of the signal is generated in the later hours when pH tends to drop.