Week 06 – Genetic Circuits Part I

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DNA Assembly

  1. Phusion High-Fidelity PCR Master Mix contains DNA polymerase, dNTPs, buffer, and Mg²⁺. The polymerase has proofreading activity (3′→5′ exonuclease), which reduces errors during DNA amplification. The dNTPs act as building blocks, while the buffer and Mg²⁺ maintain optimal conditions for enzyme activity. This is important in cloning because even small mutations can affect protein function. https://www.neb.com/en/products/e0553-phusion-high-fidelity-pcr-kit?srsltid=AfmBOooI-JWWTJ01XuZL3foWSnvq5kqQol7r8q61xRo95a6S7amAGeiH

  2. Annealing temperature depends mainly on primer Tm (melting temperature), GC content, and primer length. Tm is the temperature at which half of the DNA strands separate. Higher GC content increases Tm because GC bonds are stronger. If the temperature is too low, primers bind nonspecifically, and if it is too high, they fail to bind. https://academic.oup.com/nar/article/18/21/6409/2388653?login=false

  3. PCR and restriction digestion both generate linear DNA fragments, but they differ in approach. PCR amplifies a specific DNA region using primers and allows modification of sequences, such as adding overlaps or mutations. Restriction digestion uses enzymes to cut DNA at specific recognition sites, producing defined fragments without amplification. PCR is more flexible and useful when designing constructs or preparing fragments for Gibson assembly. Restriction digestion is more straightforward but depends on the availability of suitable restriction sites in the DNA sequence.

  4. To make sure the DNA fragments work for Gibson assembly, the main thing is that they have overlapping sequences, usually around 20–40 base pairs. These overlaps are not random, they need to match exactly between adjacent fragments so they can base-pair during the reaction. In practice, these overlaps are usually added through primer design during PCR. Another important point is using a high-fidelity polymerase, because even small mutations in the overlap region can prevent proper assembly. It’s also a good idea to check fragment size on a gel and confirm sequence accuracy before moving forward. If the overlaps are not correct or the DNA quality is poor, the assembly simply won’t work, since Gibson relies entirely on sequence homology rather than restriction sites. https://www.nature.com/articles/nmeth.1318

  5. From what I understood in the protocol, the plasmid doesn’t enter the cells through any active mechanism. The cells are first made competent using calcium chloride, which helps reduce the repulsion between the DNA and the membrane. Then during heat shock, the sudden temperature change kind of destabilizes the membrane and creates temporary openings, so the DNA can slip inside. So it’s really more of a physical process rather than something controlled by the cell. After that, the cells recover and start expressing the plasmid. This also explains why transformation efficiency depends a lot on how well the cells were prepared. https://www.sciencedirect.com/science/article/abs/pii/S0022283683802848?via%3Dihub

  6. Golden Gate Assembly is different from Gibson because it depends on restriction enzymes instead of sequence homology. It uses Type IIS enzymes like BsaI, which cut outside of their recognition site. This is useful because it lets you design specific overhangs, so different fragments can attach to each other in a defined order. What I found interesting is that digestion and ligation happen in the same reaction, so the system basically cycles between cutting and joining until the correct construct forms. Also, the recognition sites are removed during the process, so the final DNA doesn’t contain extra unwanted sequences. This makes it especially useful when assembling multiple fragments at once, since you can control how everything connects. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647