https://pages.htgaa.org/2026a/jason-ross/homework/week-06-hw-genetic-circuits-part-i/index.htmlRobot 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:Hugoen