Genetic Circuits - 1

Part - 1

1. What are some components in the Phusion High-Fidelity PCR Master Mix, and what is their purpose?

   • Phusion High-Fidelity PCR Master Mix, commonly produced by Thermo Fisher Scientific, contains a high-fidelity DNA polymerase with proofreading ability, a reaction buffer that maintains optimal conditions, Mg²⁺ ions as a cofactor, dNTPs as building blocks, and stabilizing additives. Together, these components enable accurate and efficient DNA amplification with a low error rate.

2. What are some factors that determine primer annealing temperature during PCR?

   • Primer annealing temperature in PCR is mainly determined by the melting temperature of the primers, which depends on their length and GC content. Higher GC content and longer primers increase the melting temperature, leading to a higher annealing temperature, while mismatches and low salt conditions can reduce it.

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.

   • PCR and restriction enzyme digestion both generate linear DNA fragments but differ fundamentally in approach. PCR amplifies DNA from a template using a polymerase and primers, making it ideal when starting material is limited or when sequence modifications are needed, while restriction digestion cuts existing DNA at specific sequences using enzymes, making it preferable when precise, predefined sites are available and no amplification is required.

4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

   • PCR and restriction enzyme digestion both generate linear DNA fragments, but differ fundamentally in approach. PCR amplifies DNA from a template using a polymerase and primers, making it ideal when the starting material is limited or when sequence modifications are needed, while restriction digestion cuts existing DNA at specific sequences using enzymes, making it preferable when precise, predefined sites are available, and no amplification is required.

5. How does the plasmid DNA enter the E. coli cells during transformation?

   • To ensure DNA fragments are suitable for Gibson Assembly, the sequences must be designed with overlapping ends of about 20 to 40 base pairs that are complementary between adjacent fragments. These overlaps must have appropriate melting temperatures and correct sequence alignment so that the fragments can anneal properly and be joined seamlessly.

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 works by repeatedly cycling between digestion and ligation in one reaction mixture containing DNA fragments, a Type IIS enzyme, and ligase. The enzyme cuts to create specific overhangs, fragments anneal based on complementary ends, and ligase seals them together. Because the recognition sites are eliminated after cutting, correctly assembled products accumulate over time. This enables efficient and accurate multi-fragment assembly without leaving extra sequences between parts. The method is widely used in synthetic biology for building complex constructs.