https://pages.htgaa.org/2026a/jessee-svoboda/homework/week-06-hw-genetic-circuits-part-i/index.htmlContents DNA Assembly questions Asimov Kernel DNA Assembly questions What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion DNA polymerase - a high fidelity DNA polymerase, which means that it is an enzyme that adds single nucleotides to extend a DNA chain along a template with some sort of proof-reading ability. It is used for PCR, which means it has to be thermostable. dNTPs - single nucleotide bases to be used by the polymerase to make DNA buffer - buffer is used primarily for controlling the pH of the PCR reaction, but it also includes MgCl2 which is a required co-factor for the DNA polymerase. What are some factors that determine primer annealing temperature during PCR? Primer annealing temperature is affected by the length of the primer and the GC content primarily. 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 is a method to produce many copies of a DNA sequence for which you already have a template. It requires a thermocycler, and PCR mix (thermostable DNA polymerase, dNTPs, appropriate buffer). To use it, you need to have template DNA and primers designed to bookend the sequence of interest. Restriction digests can linearize circular DNA or trim DNA sequences. It requires a heat block or incubator, the relevant restriction enzymes, and appropriate buffer. To use it, you need to have (typically a medium or high concentration amount) DNA that contains your sequence of interest already bookended by restriciton enzyme cutsites. Restriction digests can produce sticky ends or blunt ends; PCR will always produce blunt ends. Both methods will typically require some sort of purification step before further use (DNA cleaning and concentrating; gel extraction). PCR is useful when you need more of a particular sequence of DNA, when you want to make point mutations within a sequence (multi-step process), to add short sequences to the ends of the DNA sequence (such as restriction enzyme cutsites, adaptors, or overlaps). Restriction digestion is useful when you need to remove an insert from a plasmid backbone, to linearize a vector for electrophoresis or other analysis, and for restriction-digest cloning (including ensuring insert and vector have appropriate sticky ends for directional insertion). How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Ideally you would design and test in silico to ensure overlaps are appropriate. My first couple times trying Gibson assembly, i wrote it out by hand to convince myself i had done it correctly, but many molecular biology software options can now assist with this as well. You can exactly confirm your purified DNA fragments prior to Gibson assembly by sequencing them, but you can also just get a good idea of their size (which would at least tell you if you PCR’d a very different or non-specific products) by running them on a gel. How does the plasmid DNA enter the E. coli cells during transformation? During a heat shock transformation, you shock the E. coli cells with an abrupt temperature change from on ice at 0°C (or sometimes room temperature around 20°C) to 42°C. This opens pores within the cell membrane that allow DNA to enter the cells, due to prior treatment with CaCl2 to neutralize the negative charge of the DNA. 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 can be conceptualized as a cross between restriction digest cloning and Gibson Assembly. Like restriction digest cloning, restriction enzymes are used to digest both the insert and the vector to create compatible sticky ends for directional insertion. However, it uses Type IIS restriction enzymes (such as AarI) that cut outside their recognition site. Therefore with correct design, the recognition sites are removed in assembly. This allows for plasmid construction similar to Gibson assembly: design your insertion fragments and vector backbone to have compatible overhangs/overlaps with the adjacent sequences (often added during primer design in PCR), then add all fragments to the reaction mix which includes both a nuclease and a ligase for assembly. In Golden Gate assembly, the Type IIS restriction enzyme(s) find their recognition sites, cut nearby (at a pre-identified base), resulting in the designed 4-base overhangs. These overhangs can connect with matching overhangs from either the original construct or the intended adjacent fragment, which will be ligated into a closed dsDNA molecule (if the original construct is re-ligated, then the Type IIS enzyme again finds the recognition site and cuts again, thereby improving the efficiency). Figure from Addgene’s Golden Gate Cloning page. Model this assembly method with Benchling or Asimov Kernel! To compare assembly methods, I used Benchling’s Assembly Wizard tool to simulate the same plasmid construction using restriction digest, Gibson assembly, and Golden Gate assembly. My target plasmid is called “pGFP”, with a pET28a(+) backbone and an insert containing the gene for green fluorescent protein (GFP) under constitutive promoter P_LacIQ from plasmid pZE27GFP. I started by importing both pET28a(+) and pZE27GFP into Benchling from Addgene. I used Benchling’s auto-annotation tool on pET28a(+) for annotations. pZE27GFP was already annotated, but was missing the annotation for P_LacIQ, so I added an annotation from that by downloading the Genbank file from the Addgene site and using CTRL-F on the sequence to identify it in the original file. I wanted these annotations so that I knew the locations of the relevant sequences in my files for easier visual identification during the cloning simulation. Note that the GFP translation in the pZE27GFP file didn’t include the stop codon, but the stop codon was present, just not included in that translation annotation, and I was too lazy to fix this, so I just remembered that my sequence of interest included the three bases past the end of the translation annotation. Restriction Digest Opening the pZE27GFP file to the plasmid map view, I selected the Digests tool to show all single cutters on the map, and identified ones that were near the ends of goal insertion sequence (outside P_LacIQ and GFP): XhoI and HindIII.Hugoen