Week 6 HW: Genetic Circuits Part 1
Assignment: DNA Assembly
1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
- Phusion DNA Polymerase: This is the “engine.” It’s a highly thermostable enzyme that synthesizes new DNA strands. It’s “High-Fidelity” because it has $3’ \rightarrow 5’$ exonuclease activity (proofreading), making significantly fewer mistakes than standard Taq.
- dNTPs (Deoxynucleotide Triphosphates): These are the molecular building blocks (A, T, C, and G) used by the polymerase to construct the new DNA strand.
- Buffer (containing $Mg^{2+}$): Maintains the optimal pH for enzymatic activity and provides essential divalent cations. Magnesium ions act as a cofactor for the polymerase, helping it catalyze the phosphodiester bond.
- Stabilizers: Often includes detergents or proprietary chemicals to prevent the enzyme from denaturing or sticking to the tube walls during the high-heat cycles.
2. What are some factors that determine primer annealing temperature during PCR?
- Primer Length: Longer primers generally require higher temperatures to remain specific.
- GC Content: G-C pairs have three hydrogen bonds compared to the two in A-T pairs. Therefore, primers with higher GC content have higher melting temperatures ($T_m$).
- Salt Concentration: The concentration of monovalent cations (like $K^+$) in the buffer affects the stability of the DNA duplex.
- Primer Concentration: Higher concentrations can slightly shift the kinetics of annealing.
- Mismatches: If the primer isn’t a 100% match to the template, the $T_m$ will decrease.
Note: The annealing temperature ($T_a$) is usually chosen to be $3-5^\circ\text{C}$ below the $T_m$ of the primers to balance specificity and yield.
3. Compare and contrast PCR vs. Restriction Enzyme Digests.
| Feature | PCR (Polymerase Chain Reaction) | Restriction Enzyme Digest |
|---|---|---|
| Mechanism | Enzymatic synthesis of new DNA strands. | Enzymatic “cutting” of existing DNA strands. |
| Input | Template DNA + Primers + Polymerase. | Plasmid or genomic DNA + Specific Enzymes. |
| Output | Exponentially amplified linear fragments. | Linearized fragments (no amplification). |
| Customization | Very high; you define the ends via primers. | Limited to where specific “sites” (e.g., EcoRI) exist. |
| Accuracy | Risk of point mutations (minimized by Phusion). | Highly accurate sequence retention. |
When to use which?
- Use PCR when you need to add specific “overhangs” for Gibson assembly or when you have a very small amount of starting material.
- Use Restriction Digest when you are moving a large chunk of DNA from a “classic” vector that already contains the necessary sites, or when you want to avoid the risk of PCR-induced mutations in a large gene.
4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
For Gibson Assembly to work, your fragments must have homologous overlapping ends (typically 20–40 base pairs).
- For PCR: You must design your primers so that the 5’ end of the primer contains a sequence that matches the end of the adjacent fragment.
- For Digest: You must ensure the restriction site is positioned such that the resulting linearized DNA shares overlap with the next piece, or use a “Stitch PCR” on the digested fragment to add the necessary overlaps.
- Verification: Use a tool like NEB’s Gibson Assembly Designer or Benchling to simulate the “junctions” and confirm the overlaps are in the correct orientation ($5’ \rightarrow 3’$) and have a high enough $T_m$ to stay stable during the reaction.
5. How does the plasmid DNA enter the E. coli cells during transformation?
In the HTGAA lab context, we usually use Chemically Competent cells:
- Heat Shock: Cells are kept on ice with DNA, then suddenly moved to $42^\circ\text{C}$.
- Pore Formation: This temperature spike creates a pressure imbalance and temporary “pores” or thermal fluctuations in the chemically-weakened cell membrane.
- DNA Uptake: The DNA moves through these temporary pores into the cytoplasm.
- Recovery: Cells are placed back on ice and then incubated in SOC/LB media at $37^\circ\text{C}$ to “heal” the membrane and begin expressing the antibiotic resistance gene before plating.
6. Describe another assembly method in detail: Golden Gate Assembly.
Golden Gate Assembly relies on Type IIS restriction enzymes (like BsaI or BpiI). Unlike standard enzymes, these cut outside of their recognition sequence, creating custom non-palindromic 4-base overhangs.
Because the recognition site is removed during the cleavage, the reaction is “directional” and “seamless.” This allows for a “one-pot” reaction where digestion and ligation happen simultaneously in the same tube. You can assemble multiple fragments (up to 10+) in a specific order by designing unique 4-bp overlaps for each junction. It is highly efficient and leaves no “scar” sequences if designed correctly.