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 and fungal biomaterials ⚙️ Part 1: Intracellular artificial neural networks (IANNs) 🧠 1. What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? IANNs offer several advantages over traditional genetic circuits, which are governed by Boolean logic, as they can integrate multiple inputs simultaneously to produce an output. Similar to biological brains, they can process information in a more adaptive manner, as they are capable of learning from cellular environments that constantly change, thus responding faster to fluctuations in their surroundings than conventional gene-regulation systems 1. Another one of their advantages is that they significantly improve decision-making accuracy inside cells by reducing noise in gene expression 2. This way, they also enable more complex computational tasks within living cells, in turn allowing the design of highly sophisticated cellular behaviors 3. This degree of scalability and control, along with their versatility, renders IANNs particularly well-suited for numerous applications in Synthetic Biology, especially in targeted therapies and personalized medicine, where the level of fine-tuning and precision that can be achieved with a genetic circuit plays a tremendously important role 2 3.
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, which, in addition, can later be purified more easily, without impurities. 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 1.
Advanced imaging and measurement technology 🎞️ 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. My final individual project revolves around developing a lichen-based building coating that is prompted to change its color by the conditions of its environment as a means of passive heat acclimation. The biomaterial can assume two different colorations, a lighter one for hot sunny days and a darker one for days when the weather is cold and cloudy. The two colorations are mediated by two different compounds, namely the protein reflectin and the pigment eumelanin respectively. As engineering a lichen is quite challenging, especially when taking into consideration the very short time frame of the course, for my first experimental aim, I intend to design a genetic circuit that will emulate the color-changing effect of the lichen construct, although, adapted for expression in E. coli, which is simpler and easier to genetically manipulate, as proof-of-concept. After inducing the synthesis of reflectin and melanin in the bacterial system, I would like to test the responsivity and functionality of the color-shifting circuit first with a simple spectrophotometric measurement, which should be feasible given that reflectin is highly reflective when interacting with visible light, whereas eumelanin is highly absorbant. As a semi-quantitative method, I could also visualize the expression of reflectin and MelC2, an essential enzyme for the biosynthesis of eumelanin, by running an SDS-PAGE protein electrophoresis and, subsequently, Western blotting. To this end, I have attached a C-terminal tag to both proteins, more specifically a 6x-His tag to reflectin and a FLAG tag to MelC2, which will allow me to use anti-His and anti-FLAG antibodies to examine the expression of each protein in the Western blot. Alternatively, for a more precise quantification, I could purify the proteins and analyze them through mass spectrometry. This way, I could obtain both measurements about each protein’s level of expression, but also useful data both their respective sequences to verify that the proteins are synthesized as anticipated in my system. Another advantage of mass spectrometry-facilitated analysis is that, unlike SDS-PAGE and Western blots, it can be utilized to investigate other biomolecules too. Therefore, instead of indirectly quantifying eumelanin production through measurement of MelC2 expression, directly monitoring how much eumelanin has been generated would be possible. Going a step back, before the induction of protein expression, I would like to have already sequenced my final assembled DNA constructs, to validate that the plasmids constructed in the lab harbor the same sequence as the ones theoretically designed and display the anticipated functions. Lastly, apart from sequencing the plasmids before the bacterial transformation, it is prudent to do the same with colonies identified as positive transformants through the selection process by extracting their plasmids and isolating the insert containing the genetic cassette(s) responsible for the different colorations. The isolation of the insert can be easily achieved either by performing a strategic restriction digestion or, in case the plasmid lacks restriction sites, by amplifying the insert through PCR and, subsequently, visualizing the result of the reaction with an agarose gel electrophoresis in both scenarios.
Bioproduction and cloud labs 🥼 Part A: The 1,536 Pixel Artwork Canvas | Collective artwork Contribute at least one pixel to the global artwork experiment before the editing ends on Sunday 19/04 at 11.59pm EST. A personalized URL was sent to the email address associated with your Discourse account, and you can discuss the artwork on the Discourse. Make a note on your HTGAA webpages including: what you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”) what you liked about the project, and what about this collaborative art experiment could be made better for next year. What survived of my main contributions to the bioart project are initiating and adding several pixels in the DNA double helix positioned in the left part of the bottom right plate. I also painted some of the Electra2 blue pixels in the background of the same plate (Figure 11.1).