Homework
Weekly homework submissions:
PRE-LECTURE ABOUT CLASS 2 SLIDES ABOUT PROFESSOR JACOBSON -Question 1 When biological polymerase copies DNA, it makes about 1 mistake per million base pairs (1:10^6).Since the human genome has around 3.2 billion base pairs, that error rate would mean every time one of my cells divides, it would introduce over 3,000 mistakes if there weren’t any correction mechanisms. There’s a 3’-5’ exonuclease that catches and removes errors during DNA synthesis, and then the MutS repair system acts as a backup to fix any mismatches that slipped through afterward. Together, these mechanisms keep my genetic information stable across cell divisions.
Week 1 HW: Principles and Practices
- Biological engineering application: The development of various therapies in which they employ stem cells in Peru, to treat neurodegenerative diseases and chronic diseases. Along with this the appropriate regulations for this type of therapy. -> Why I chose this application: Because in Peru there is an increase in the development of various clinical therapies for diseases that have been very expensive to access, so the emergence of stem cell applications are recent. This is also due to a problem, this is more than anything focused on the little regulation on this type of treatment, which limits the creation of more trained centers, generating a delay in the access of new therapies and research.
Paper: Accelerated high-throughput imaging and phenotyping system for small organisms Link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0287739 This paper details the creation of a high-throughput experimentation (HTE) platform built around duckweeds — specifically Lemna minor, a tiny aquatic plant with applications in bioremediation and biofuel research. To run large-scale evolutionary ecology experiments, the team combined an Opentrons OT-2 liquid handling robot with a custom autonomous imaging system, creating a pipeline capable of operating at a scale that would be practically impossible by hand. The central engineering challenge was that standard liquid handling robots are designed to work with, unsurprisingly, liquids. Duckweeds are solid floating plant fronds, which meant the OT-2 needed to be rethought for a very different kind of material. The researchers solved this by replacing the standard pipette tips on the OT-2’s P300 pipette heads with commercial inoculation loops. These loops exploit capillary action to gently lift individual fronds from the water’s surface, allowing the robot to pick and place solid biological matter with the same reliability it would otherwise bring to liquid transfers. This seemingly simple hardware modification had enormous practical consequences. By enabling automated handling of the plants, the team was able to design an experiment encompassing 6,000 individual microcosms spread across 2,000 distinct combinations of nutrients and microbes — a scale of experimental complexity that manual pipetting and plant placement could never realistically achieve, given how tedious and error-prone working with tiny floating organisms at high volume would be for human researchers.
Week 4 HW: Protein Design Part 1
Conceptual Questions — Q1: 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) First, we need to calculate the number of moles and multiply by Avogadro’s number (NA=6.022×1023 mol−1). An amino acid has an average mass of ~100 Daltons (Da), which is roughly equivalent to 100 g/mol. Meat is mostly protein (~20% of its weight is protein). → 500 g of meat contains approximately 100 g of protein. Since 1 mole of amino acids weighs ~100 g, there are ~1 mole of amino acids in 100 g of protein. → 1 mole is equivalent to 6.022 × 1023 molecules (Avogadro’s number). So you consume approximately 6 × 1023 amino acids in 500 g of meat.
Week 6 HW: GENETIC CIRCUITS PART I: ASSEMBLY TECHNOLOGIES
- What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The Phusion Master Mix is basically a ready-to-use mix that makes setting up PCR way easier since everything is already in it. The main component is the Phusion High-Fidelity DNA Polymerase, which is the enzyme that actually copies the DNA. What makes it special is that it catches and fixes mistakes as it goes, giving you really accurate amplification. It also has the four dNTPs which are the building blocks the polymerase uses to build new DNA strands. There’s also a reaction buffer that keeps the pH and salt conditions stable so the enzyme works properly. 2.What are some factors that determine primer annealing temperature during PCR? The annealing temperature matters a lot because if it’s too low, your primers bind nonspecifically and you get messy results, and if it’s too high, they won’t bind at all. The most important factor is the melting temperature (Tm) of your primers — the annealing temperature is usually set about 3–5°C below the lower Tm of the two primers. Primer length plays into this too since longer primers have higher Tm values. GC content is another big one — G-C pairs have three hydrogen bonds instead of two, so GC-rich primers are more stable and need higher temperatures to melt. 3.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. Both methods can give you linear DNA fragments, but they work pretty differently. PCR uses primers, a polymerase, and thermal cycling to amplify a specific region. The big advantage is flexibility — you can design primers to add any sequence you want to the ends of your fragment, like overlaps for Gibson assembly. The downside is there’s some risk of mutations, though high-fidelity polymerases like Phusion make this pretty minimal. Restriction digestion, on the other hand, cuts DNA at specific recognition sequences using enzymes, and it’s done at a constant temperature. 4.How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Gibson assembly works by joining fragments that share overlapping sequences at their ends (usually around 20–40 bp), so you have to make sure those overlaps are designed correctly. For PCR fragments, you add the overlap sequences directly into the 5’ tails of your primers — so the forward primer of one fragment carries the end of the previous fragment, and so on. For restriction-digested fragments, you need to check that the ends left by the enzyme line up with the overlap region of the adjacent fragment. The best way to check everything is to model the assembly in software like Benchling before you even start — you can simulate how all the fragments will come together and catch any design errors early. Basically, if the end of fragment A perfectly matches the beginning of fragment B, you’re good. 5.How does the plasmid DNA enter the E. coli cells during transformation? E. coli cells don’t naturally take up DNA, so you have to make them “competent” first. With chemically competent cells, they’re treated with CaCl₂, which neutralizes the negative charges on both the DNA and the cell membrane. Then you do a heat shock which temporarily disrupts the membrane and lets the plasmid get in. With electrocompetent cells, it’s a bit different: a brief electroporation creates tiny pores in the membrane that the DNA can pass through. Both methods are essentially creating a temporary opening in the membrane for the DNA to enter. 6.Describe another assembly method in detail (such as Golden Gate Assembly) Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online). Golden Gate Assembly is a really elegant method that lets you assemble multiple DNA fragments in a single reaction. It uses Type IIS restriction enzymes — like BsaI — which are special because they cut outside of their recognition sequence. So you can engineer your fragments so that after the enzyme cuts, it leaves behind a specific 4-bp overhang that you designed, and the recognition site itself gets removed. When you run the digestion and ligation at the same time in one tube, the correctly assembled fragments stick together through their matching overhangs and get ligated. Any fragments that aren’t assembled correctly still have the recognition site, so they just get cut again.
Week 2 HW: DNA READ, WRITE, & EDIT
DNA Design Challenge Protein: GFP (Green Fluorescent Protein) Reason: Because GFP is commonly used as a biological marker to visualize various cellular processes due to its green fluorescence. sp|P42212|GFP_AEQVI Green fluorescent protein OS=Aequorea victoria OX=6100 GN=GFP PE=1 SV=1 MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence GFP DNA >ATGTCCAAGGGTGAGGAGCTGTTTACCGGCGTGGTTCCGATTCTTGTGGAATTAGACGGCGATGTCAACGGCCACTTCTCCGTTTCT GGCGAGGGCGAGGGAGGCGACGCCACGTATGGCAAATTGACCCTGAAGTTTATTTGCACGACCGGAAAATTGCCTGTACCGTGGCCCACACTTTGGT CACTACCGTTATCAATGTTTCTCGCTATCCGGACCACATGAAGCAGCATGACTTCTTTAAAAGTGCAATGCCCGAGGGTTATGTTCAAGAGCGGACCA TCTTTTTTAAAAGACGACGGCAACTACAAGACGCGCGAGGTGAAGTTCGAGGGCGACACGCTGGTGAATCGGATTGAGTTAAAAGGAATTGACTTTAA AAGATGACGGCAACATCCTTGGACATAAGTTAGAGTACAATTATAATTCAAACCACGTGTACATCATGGCCGACAAACAAAAAAACGGCATCAAGGTA AACTTTAAAATTAGACATAATATCGAGGATGGCAGTGTTCAATTAGCCGACCATTACCAACAGAACACCGATAGGCGGACGGTCCTGTATTACCTGAC AACCATTACCTTAGCACGCAGTCTGCACTGTCCAAGGACCCAAATGAGAAACGAGGACCATATGGTGTTGCTAGAGTTCGTTACCGCAGCAGGAATAAC Codon optimization The selected organism: Escherichia coli (E. coli) Reason: This is because E. coli is a common model organism for the production of recombinant proteins due to its speed, low cost and ease of manipulation.