Homework
Weekly homework submissions:
Principles and practices 💼 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. Purification of enzymes for natural pigment synthesis facilitated by microalgal cell wall release
DNA read, write, and edit 🧬 Part 1: Benchling and in-silico gel art The genome of the λ-phage was imported and virtually digested with the following restriction endonucleases: EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, and SalI before being visualized on Benchling’s agarose gel simulator (Figure 2.1).
Lab automation 🦾 Python script for Opentrons artwork Generate an artistic design using Ronan’s GUI. 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. You may use AI assistance for this coding — Google Gemini is integrated into Colab (see the stylized star bottom center); it will do a good job writing functional Python, while you probably need to take charge of the art concept. If you use AI to help complete this homework or lab, document how you used AI and which models made contributions. Consistent with this week’s highly automated and digitized theme, for this assignment, I drew inspiration from an image popularized by the Internet, KC Green’s web comic strip “On Fire”, which, in 2014, became a famous -and my personal favorite- online meme (Figure 3.1). As many other people from all over the world, I deeply relate to this meme, which, I feel, accurately describes my life.
Protein design-Part I 💻 Part 1: Conceptual questions Answer any nine of the following questions from Shuguang Zhang: (i.e. you can select two to skip) 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.) Depending on the type of meat, as well as the manner it is processed prior to consumption, 500g of meat contain approximately 100 - 130g of protein. Assuming that this protein consists entirely of amino acids (meaning, excluding metal ions, such as iron or zinc, which can be found bound to protein molecules, or glycans and other moieties added to proteins through post-translational modifications), then 100-130g of amino acids = 6.02 - 7.83x1025Da approximately. Therefore, if the molecular weight of one amino acid is on average ~100Da, then 500g of meat contain (6.02 - 7.83x1025Da)/100Da = 6.02 - 7.83x1023 amino acid molecules.
Protein design-Part II 💻 Part 1: 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.
Genetic circuits-Part I: Assembly technologies 🧩 DNA Assembly Answer these questions about the protocol in this week’s lab: 1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The components in the Phusion High-Fidelity PCR Master Mix, along with their purpose, are the following:
Genetic circuits-Part II: Neuromorphic circuits 🧠
Cell-free systems 🧪 General homework questions 1. 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. Compared to conventional in vivo methods, cell-free protein synthesis provides modularity and substantially higher experimental control, as all the system’s components can be readily added or removed, especially when the strategy employed is to separately produce or extract each cellular element required for the process and then combine them all together into a single reaction. Cell-free systems also offer the potential for precise control over reaction conditions, such as pH and ion concentration, while being more flexible and versatile since they allow the expression of proteins deleterious to living cells, support the integration of non-natural and non-canonical amino acids into peptide backbones, and are compatible with diverse DNA templates (linear or plasmid). Additionally, they eliminate constraints imposed by the existence of living cells. For instance, unlike traditional cell cultures, they do not need any monitoring, cultivating, or other interventions aimed at preservation, nor are they susceptible to issues of cell viability, growth limits, or stress responses. Similarly, since the cell-free apparatus exists outside of the context of a cellular platform, there are no cell-membrane barriers, facilitating access to biochemical reactions, while, at the same time, there is no interference or competition from other metabolic procedures or regulatory signals, enabling all the available resources to be channeled towards the synthesis of the desired protein. The absence of living cells can be translated into abolishing the need for cloning and cellular transformation as well, which, in turn, ensures safer handling, as no genetically modified organisms are involved in cell-free protein production. More generally, one of the method’s most significant advantages is that it is a highly efficient technique for rapid protein synthesis that can also withstand being transferred across larger distances for longer periods of time, as the entire system can be easily freeze-dried and stored for later use.
Advanced imaging and measurement technology 🎞️ 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). 1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight? eGFP amino acid sequence with C-terminal linker and 6x-His tag