Week 6 HW: Genetic Circuits Part I

DNA Assembly

Answer these questions about the protocol in this week’s lab: What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

Typical components include:

  • Phusion DNA Polymerase A high-fidelity enzyme that synthesizes new DNA strands with very low error rates (proofreading activity → fewer mutations).
  • dNTPs (deoxynucleotide triphosphates) Building blocks (A, T, G, C) used to create new DNA strands.
  • Reaction Buffer (HF buffer) Maintains optimal pH and salt conditions for enzyme activity and fidelity.
  • Mg²⁺ ions (magnesium chloride) Essential cofactor for polymerase activity; affects enzyme efficiency and specificity.
  • Stabilizers (sometimes included) Help maintain enzyme stability during thermal cycling.

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

Annealing temperature is critical for specificity. It depends on:

Primer melting temperature (Tm) Main determinant; annealing temperature is usually ~2–5°C below the lowest Tm. *Primer length Longer primers → higher Tm. GC content (40–60% ideal) More G/C = stronger binding → higher Tm. Sequence composition GC-rich regions bind more tightly than AT-rich ones. Salt concentration in buffer Higher salt stabilizes DNA duplexes → increases effective Tm. Primer mismatches Intentional mismatches (like your mutation) can slightly lower binding strength.

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

Protocol:

  • Uses primers + polymerase to amplify a specific DNA region
  • Requires thermocycling (denature → anneal → extend)

Advantages:

  • Can introduce mutations (e.g., your chromophore changes)
  • No need for restriction sites
  • Very flexible and precise

Limitations:

  • Can introduce errors (though minimized with high-fidelity enzymes)
  • Requires careful primer design
Restriction Enzyme Digest

Protocol:

  • Uses enzymes that cut DNA at specific recognition sites
  • Produces sticky or blunt ends

Advantages:

  • Highly specific and reproducible
  • No amplification errors

Limitations:

  • Requires existing restriction sites
  • Leaves “scars” (extra sequences)
  • Less flexible for mutations

When one wants to mutate Dna and doesn’t have suitable restriction sites, one uses PCR. However, if one wants a simple, clean cloning and suitanle restriction sites already exist, one uses Restriction Enzyme Digest.

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

To ensure compatibility:

  • Design overlapping regions (20–40 bp): Adjacent fragments must share identical sequences.
  • Correct orientation (5′ → 3′): Overlaps must align properly for assembly.
  • Accurate primer design: Overhangs must match the adjacent fragment exactly.
  • High-quality DNA fragments: Clean PCR products (no contaminants or leftover template).
  • Remove template plasmid (DpnI step): Prevents background colonies from unmutated DNA.
  • Verify fragment sizes (gel electrophoresis): Confirms correct amplification.

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

  1. Cells are made chemically competent (membrane destabilized).
  2. DNA is added and incubated on ice → DNA associates with membrane.
  3. Heat shock (42°C) creates temporary pores in the membrane.
  4. DNA enters the cell through these pores (diffusion-driven).
  5. Cells recover in SOC media and begin expressing the plasmid (e.g., antibiotic resistance).

Describe another assembly method in detail (such as Golden Gate Assembly)

Golden Gate Assembly uses Type IIS restriction enzymes (e.g., BsaI) that cut DNA outside of their recognition sequence, generating custom overhangs. These overhangs allow multiple DNA fragments to be assembled in a specific order in a single reaction. Unlike Gibson Assembly, Golden Gate uses a cut-and-ligate mechanism, where restriction enzyme digestion and ligation occur simultaneously in a thermocycling reaction. Because the recognition sites are removed during assembly, the final construct is scarless. This method is highly efficient for assembling multiple fragments (e.g., synthetic biology constructs). It is especially useful for modular cloning systems. However, it requires that internal restriction sites be removed beforehand.

Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

  • Golden Gate Assembbly Method needs to be added

Model this assembly method with Benchling or Asimov Kernel!

N/A because we don’t have access.