Homework
Weekly homework submissions:
Hello 1 World ↩︎
Week 1 HW: Principles and Practices
A glorious space phage (with artistic license lol) Week 1 Biological Engineering Application Governance Exercise I’m interested in developing phage chassis capable of targeting bacteria that commonly cause infections during spaceflight. I’m interested in developing these phage chassis because:
Week 2 HW: DNA Read, Write, and Edit
Part 0: Basics of Gel Electrophoresis Per instructions for this part, I attended the 02/10 lecture and 02/11. Additionally I attended all 3 Bootcamp sessions. Part 1: Benchling & In-silico Gel Art Make a free account at benchling.com Benchling Account Creation Confirmation
The Power of Lab Automation Assignment: Python Script for Opentrons Artwork 0: Attended this week’s recitation and reviewed the lab information on programming Opentrons 1: Generated an artistic design using Ronan’s Opentrons GUI 1 2: Artistic Design Python Script: See script in URL below: https://colab.research.google.com/drive/1-pgSJt_aF9MydtG0szxz2YKoogNRLRhH#scrollTo=PsOgJ2DndZzt 3: Listing my sfgfp point coordinates from Ronan’s Opentrons GUI below (the shape is a rightward-facing green arrow): [(6.6,11), (8.8,11), (11,11), (8.8,8.8), (11,8.8), (13.2,8.8), (11,6.6), (13.2,6.6), (15.4,6.6), (13.2,4.4), (15.4,4.4), (17.6,4.4), (15.4,2.2), (17.6,2.2), (19.8,2.2), (17.6,0), (19.8,0), (22,0), (-22,-2.2), (-19.8,-2.2), (-17.6,-2.2), (-15.4,-2.2), (-13.2,-2.2), (-11,-2.2), (-8.8,-2.2), (-6.6,-2.2), (-4.4,-2.2), (-2.2,-2.2), (0,-2.2), (2.2,-2.2), (4.4,-2.2), (6.6,-2.2), (8.8,-2.2), (11,-2.2), (13.2,-2.2), (15.4,-2.2), (17.6,-2.2), (19.8,-2.2), (22,-2.2), (24.2,-2.2), (-22,-4.4), (-19.8,-4.4), (-17.6,-4.4), (-15.4,-4.4), (-13.2,-4.4), (-11,-4.4), (-8.8,-4.4), (-6.6,-4.4), (-4.4,-4.4), (-2.2,-4.4), (0,-4.4), (2.2,-4.4), (4.4,-4.4), (6.6,-4.4), (8.8,-4.4), (11,-4.4), (13.2,-4.4), (15.4,-4.4), (17.6,-4.4), (19.8,-4.4), (22,-4.4), (24.2,-4.4), (26.4,-4.4), (-22,-6.6), (-19.8,-6.6), (-17.6,-6.6), (-15.4,-6.6), (-13.2,-6.6), (-11,-6.6), (-8.8,-6.6), (-6.6,-6.6), (-4.4,-6.6), (-2.2,-6.6), (0,-6.6), (2.2,-6.6), (4.4,-6.6), (6.6,-6.6), (8.8,-6.6), (11,-6.6), (13.2,-6.6), (15.4,-6.6), (17.6,-6.6), (19.8,-6.6), (22,-6.6), (24.2,-6.6), (17.6,-8.8), (19.8,-8.8), (22,-8.8), (15.4,-11), (17.6,-11), (19.8,-11), (13.2,-13.2), (15.4,-13.2), (17.6,-13.2), (11,-15.4), (13.2,-15.4), (15.4,-15.4), (8.8,-17.6), (11,-17.6), (13.2,-17.6), (6.6,-19.8), (8.8,-19.8), (11,-19.8)]
Week 4 HW: Protein Design Part 1
South American Rattlesnakes (Crotalus durissus terrificus) with Crotamine protein 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) I intake approx. 5 * 1023 Daltons of amino acids when ingesting 500 grams of meat. This is based off results indicating I ingest approx. 1021 Daltons of amino acids when ingesting 1 gram of meat Why do humans eat beef but do not become a cow, eat fish but do not become fish? Humans eat beef but don’t become cattle, and eat fish but don’t become fish because genetic information from the lifeform being ingested isn’t transferred wholesale. Much of the genetic material being eaten is broken down during digestion and more importantly a human beings’ cells follow instructions derived from their DNA. Human beings’ cells utilize amino acids from the lifeform being ingestion, but perform this utilization according to specific genetic instructions. The lifeform being ingested and its amino acids are the raw materials the cell uses for various means. Why are there only 20 natural amino acids? There are several broad reasons why there are only 20 standard natural amino acids. The first reason is that early on in the history of evolution, this group of amino acids became more or less ’locked in’, meaning that once the basic relationship between three letter codons and these 20 standard natural amino acids became widely distributed across the kingdom of life, it becamde too risky/dangerous from an evolutionary standpoint to alter this core set. Another reason is that the group of 20 gives enough range in structure and chemistry to build a large chunk of what evolution or directed evolution might desire. The other reasons seem to amount to various types of evolutionary trade-offs. Adding more than 20 amino acids to this standard set would add additional, potentially unwanted complexity, while decreasing the number of amino acids in the set might lead to issues with a lack of uniqueness with amino acids side chain sharing, which would in turn limit the functional flexibility of amino acids to do things like fold precisely. Can you make other non-natural amino acids? Design some new amino acids. Yes you can. My attempts to design some new amino acids usng SwissSideChain and the Cryo-EM structure of Receptor Tyrosine Kinase ROS1 PDB file in PyMol (open-source) are shown below: Attempt at creating a non-natural amino acid residue mutation of Tyrosine Kinase ROS1 using cyclohexanecarboxylic acid
Week 5 HW: Protein Design Part 2
Using AlphaFold for Protein Optimization Part A: SOD1 Binder Peptide Design Part 1: Generate Binders with PepMLM Retrieved human SOD1 sequence via UniProt (see photo below). Introduced A4V mutation via Gemini prompt (see sequence below). Human SOD1 sequence (A4V mutation not added) Human SOD1 sequence (A4V mutation added)
Week 6 HW: Genetic Circuits Part 1
Robot Crafting Genetic Circuit (Stylized) DNA Assembly What are some components in the Phusion High-Fidelity (HF) PCR Master Mix and what is their purpose? HF DNA Polymerase: This is the enzyme responsible for copying DNA as it moves from the 5’ to the 3’ position across the DNA Deoxynucleotide triphosphates (dNTPs): These are the DNA molecular building blocks, consisting of Adenine (A), Thymine (T), Cytosine (C), and Guanine (G) variants HF Buffer: This consists of magnesium chloride, which is salt added to the reaction. It matters because it dissolves into Mg²⁺, which helps nucleotides bond during the reaction What are some factors that determine primer annealing temperature during PCR? Some factors that determine primer annealing tempeature during PCR include: Primer lengths Primer melting tempratures GC content/sequence content Buffer components 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. PCR: PCR creates new linear DNA fragments by via enzymatic amplification of a given region nth number of times. The PCR protocol essentially consists of setting up reaction mixes, denaturating the DNA into single strends, annealing so primers can anneal to specific complementary sequences, extension so the polymerase can syntehsize a new strand, and then repeating this as many times as neccessary. This method might be more useful when there is a specific fragment of DNA one wants to amplify for further use. Restriction Enzyme Digests: Restriction Enzyme Digests create new linear DNA fragments by cutting DNA at specific points/recognition sites. The Restriction Enzyme Digest protocol consists of setting up a reaction mix, incubation, and then stopping the reaction. This method might be more useful when there is a specific fragment of DNA one wants to isolate for further analysis. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? You can ensure the DNA sequences have appropriate 5’ –> 3’ orientation with corresponding overlaps. Fragments salso need to cover the relevant region for cloning, and also need to be inserted at the appropriate molar ratio relative to the plasmid backbone (vector). This is usually a 2:1 ratio. How does the plasmid DNA enter the E. coli cells during transformation? The plasmid DNA enters the E. coli either via heat shock (temperature change) or electroporation (high electrical voltage). Both methods shock the E. coli cell, causing its cell membrane to open for the plasmid DNA to enter. Describe another assembly method in detail (such as Golden Gate Assembly) DNA topoisomerase I (TOPO) Cloning: TOPO cloning’s traditionally used, as it’s a fast, reliable method for cloning products from PCR for later sequencing, etc. The first step in TOPO cloning is generating an insert with Taq polymerase via PCR. This creates inserts with an A-overhang, which can then help address the second step. The second step is to combine this PCR product with the TOPO vector. This is usually done for a couple of minutes. The insert’s 5’ OH/hydroxyl interacts with the TOPO DNA at its end, and as part of this process A and T base pairing occurs between the respective insert and the vector . Then the TOPO religates the strangs and dissociates, creating a closed circular plasmid with the given insert. See diagrams below:
Week 7 HW: Genetic Circuits Part 2
Genetic Circuits Part 2 Assignment Part 1: Intracellular Artificial Neural Networks (IANNs) Unlike traditional genetic circuits, IANNs are analog, and as such correspond more closely to the nature of biological systems (i.e, we’re not always looking for strict 0/1 binary logic, sometimes we’re looking to establish control across a range of values or space/time). This analog nature means they are more responsive, efficient, and biocompatible.
Homework Part A: General and Lecturer-Specific Questions Cell-Free Systems 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. Cell-free systems allow for a broader range of potential chemistries than those given to us from natural biology, expanding flexibility. Cell-free protein synthesis also allows for greater control over experimental variables because the entire protein expression construct is designed from scratch (i.e., we have the opportunity to bypass a lot of the compleity of natural cells). Cell-free expression is more beneficial than cell production if you want to rapidly protoype gene pathways and if you want an expression mechanism that’s more amenable to consistent, predictable modeling and analysis. Describe the main components of a cell-free expression system and explain the role of each component. The main components of a cell-free expression system are (based on elements described in this hyperlink 1): DNA template: Genetic code to begin Tx/Tl process Ribosomes: Assembling amino acids into polypeptides Enzymes: Catalyzing certain important chemical reactions necessary for the appropriate functioning of that cell-free expression system (ex. transcription and translation, energy generation) Amino Acids: The core chemical building blocks of the proteins the cell-free expression system will express Polymerases: Synthesizing DNA and RNA 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 Energy provision regeneration is critical in cell-free systems because cell-free systems don’t consume enzymes to produce energy. They also need external energy sources to remove waste products. A workaround might be to have analogous enzymatic reactions (possibly based off shared common charges) within the cell-free system to produce energy Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. Prokaryotic cell-free expression systems allow for the colocation of transcription and translation. This might work well for proteins that need to be produced at high volume, like an industrial protease prtoein. Eukaryotic cell-free expression systems allow for more complex proteins to be built due to their nuclei. This might work well for the production of more advanced/technically complex proteins, like rabbit serum albumin. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup. In a manner similar to Shuguang Zhang ‘molecular glove’ experiment, I’d try to essentially coat and/or surround the the membrane protein with hydrophilic proteins to attract and/or absorb water in the cell-free environment, so the membrane protein can incorporate into the liposome 2. Challenges might include appropriate hydrophilic concentrations (which might be discerned via calculations or trial and error) or bonding between the hydrophilic proteins and the membrane proteins. This might be mitigated and/or the amount of error reduced through the use of computaitonal modeling and simulation tools like AlphaFold Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each. Suboptimal Ribosome Function: Examine ribosome mRNA transcription processes and modify as necessary Suboptimal Transcription: Examine tRNAs for coding errors/misreads or inappropriate expression levels and modify as necessary Suboptimal External Communication (i.e., yields cannot properly exit system at desired levels): Examine and modify membrane channel functionality as necessary Supporting prompts for this section listed below:
Week 10 HW: Advanced Imaging & Measurement Technology
Waters Corporation Mass Spectrometer Homework: Final Project For your final project: Please identify at least one (ideally many) aspect(s) of your project that you will measure. Lysis Rate Efficiency of Plating Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. Lysis Rate: This measures the rate at which the mutated m. smegma mycobacteriophage lyses or destroys bacteria. This would be measured in a wet lab setting by comparing percentages of bacteria across a control and another plate that has been exposed to a mutated form of m. smegma mycobacteriophage Efficiency of Plating: This measures the rate at which the mutated m. smegma mycobacteriophage can begin initiating a host infection. Believe this would also be measured in a wet lab setting by comparing percentages of bacteria across a control and another plate that has been exposed to a mutated form of m. smegma mycobacteriophage What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail. Lysis Rate: I’d likely use a microplate reader as part of a wet lab extension of the final project Efficiency of Plating: I’d use a plauqe assay as part of a wet lab extension of the final project Supporting prompts for this section listed below:
Week 11 HW: Bioproduction and Cloud Labs
Part 1: Global Pixel Artwork Cloud Lab Contribution Made the following contributions to the Global Pixel Artwork Cloud Lab (see screenshots below) Global Pixel Artwork Contributions (see above). Edited 4 pixels in the upper right hand corner of the image (changed them to sfGFP)
Week 12 HW: Bioproduction & Cloud Labs Part 2
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork Contribute at least one pixel to this global artowrk experiment before the editing ends on Sunday 4/19 at 11:59 PM EST. Contributed 4 pixels to the global artwork experiment on Saturday 4/18 Make a note on your HTGAA webpages including:
Week 13 HW: Scaling Health Innovation
Master Mix Concentrations See test Master Mix Concentrations below: