Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Lab Documentation Pipetting Lab Objective: Practice accurate pipetting techniques while preparing bacterial cultures and media for in-vitro experiments. Procedure: Selected appropriate pipettes and tips for working with bacterial suspensions. Measured and transferred culture media and bacterial samples. Mixed bacterial suspensions gently to avoid damaging cells. Changed tips between samples to prevent cross-contamination. Challenges and Fixes:
Week-02-HW:-dna-read-write-and-edit
Insulin gene information 1. First, I created an account on Benchling, after which I inserted the Lambda sequence and added the restriction digestive enzymes. By combining them, I obtained the following result: 2. Unfortunately, I did not have access to a laboratory equipped with all the necessary materials to perform the experiment. 3.1 The human insulin (INS) gene is located on chromosome 11 and encodes a precursor protein called preproinsulin, which contains 110 amino acids. This precursor undergoes post-translational processing to produce the active insulin hormone, consisting of two peptide chains (A and B chains) connected by disulfide bonds.
Post-Lab Question 1: Published Paper Description Automated High-Throughput DNA Assembly using Opentrons For this assignment, I researched how the Opentrons OT-2 is utilized to automate Golden Gate Assembly for synthetic biology applications. A prime example of this is the widespread use of the OT-2 in laboratories to assemble combinatorial genetic libraries. Instead of researchers manually pipetting thousands of small-volume reactions—which is highly prone to human error and fatigue—the Opentrons robot is programmed to precisely distribute vector backbones, inserts, buffers, and enzymes into 96-well or 384-well plates.
Week-04-HW:Protein Design Part I
Part I of Protein Design How many molecules of amino acids do you take with a piece of 500 grams of meat? Meat is approximately 20% protein. • 500g $\times$ 0.20 = 100g of protein. • Average amino acid weight $\approx$ 100 Daltons. • 100g / 100 g/mol = 1 mole of amino acids. • You consume approximately $6.022 \times 10^{23}$ molecules of amino acids. Why do humans eat beef but do not become a cow, or eat fish but do not become fish? During digestion, enzymes break down foreign proteins into individual amino acids. Our cells then use our own DNA blueprint to reassemble those amino acids into human proteins. We use the same “bricks” to build a different “house.” Why are there only 20 natural amino acids? This is an “evolutionary frozen accident.” These 20 provided enough chemical variety (charge, size, polarity) for early life to survive and fold into functional shapes. Once life started using them, it became too complex to change the “standard.” Can you make other non-natural amino acids? Design some. Yes, through expanded genetic code technology. • Design Example: Photo-Leucine. It is a leucine analog that, when hit by UV light, forms a covalent bond with nearby molecules. This allows scientists to “freeze” protein-protein interactions in living bacteria. Where did amino acids come from before enzymes and life? They formed through abiotic synthesis. Experiments (Miller-Urey) showed that lightning and heat acting on primitive gases (ammonia, methane) can create amino acids. They have also been found on meteorites, suggesting they can form in space. If you make an $\alpha$-helix using D-amino acids, what handedness would you expect? Natural L-amino acids form right-handed $\alpha$-helices. D-amino acids would form a left-handed helix due to the mirrored orientation of the side chains. Can you discover additional helices in proteins? Yes. Beyond the common $\alpha$-helix, there are $3_{10}$ helices (tighter) and $\pi$-helices (wider). Why are most molecular helices right-handed? Since life uses L-amino acids, the right-handed twist is the most energetically stable configuration, as it minimizes physical clashing (steric hindrance) between the side chains and the protein backbone. Why do $\beta$-sheets tend to aggregate? What is the driving force? $\beta$-sheets have “sticky” edges with exposed hydrogen bonds. The driving force is the Hydrophobic Effect; “greasy” hydrophobic side chains want to clump together to avoid water, snapping the sheets together like magnets. Part B. Protein Analysis (IL-10) Protein Selection and Description • Protein Selected: Interleukin-10 (IL-10). • Selection Rationale: I selected IL-10 because it is a critical anti-inflammatory cytokine. It is currently at the center of cutting-edge therapeutic research where genetically modified bacteria (like Lactococcus lactis) are engineered to secrete IL-10 directly in the human gut to treat inflammatory bowel diseases (IBD) like Crohn’s disease.