Week 6 HW: GENETIC CIRCUITS PART I: ASSEMBLY TECHNOLOGIES

1. 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.