Week 6 HW: Genetic Circuits Part I

Assignment: DNA Assembly

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

  • Phusion DNA Polymerase: high-fidelity polymerase with 3’→5’ proofreading activity, so it corrects errors during extension. Much lower error rate than standard Taq.
  • dNTPs (dATP, dTTP, dCTP, dGTP): the nucleotide building blocks the polymerase adds to the growing strand.
  • MgCl₂: magnesium ions are an essential cofactor for polymerase function. Concentration affects stringency.
  • Reaction buffer: maintains optimal pH and salt conditions across the three PCR temperature steps.

The user adds template DNA and two primers (forward and reverse) to complete the reaction.

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

  • Primer length: longer primers form more hydrogen bonds with the template, raising Tm. Standard range is 18-22 bases. Too short risks nonspecific binding.
  • GC content: G-C pairs have 3 hydrogen bonds vs A-T’s 2, so GC-rich primers bind tighter and have higher Tm. Aim for 40-60%.
  • Mismatches: if the primer doesn’t perfectly match the template (e.g. when introducing a deliberate mutation), binding is weaker and you may need to lower annealing temperature or lengthen the primer.
  • Salt concentration: cations stabilize the primer-template duplex. Higher salt raises effective annealing temperature.
  • Primer pair matching: both primers should have similar Tm values (52-58°C range). Mismatched Tm between primers makes the reaction inefficient.

3. Compare and contrast PCR and restriction enzyme digests for creating linear DNA fragments.

PCR uses synthetic primers to define fragment boundaries, then amplifies the region between them. Full control over where you cut. Can also introduce mutations via primer mismatches and add overlaps for Gibson Assembly. Requires a thermocycler and careful primer design.

Restriction enzyme digests use enzymes that recognize and cut specific short sequences (4-8 bp). Simpler, cheaper, faster, but you’re limited to wherever those recognition sites naturally occur. If a site appears in an unwanted location, you get extra fragments.

Use restriction digests when convenient cut sites already exist at the right positions. Simple and cheap for basic two-part cloning.

Use PCR when you need custom fragment boundaries, want to introduce mutations, or need overlapping fragments for Gibson Assembly.

Key output difference: restriction digests give sticky or blunt ends defined by the enzyme. PCR gives blunt ends by default, but primers can be designed with 5’ tails to add any desired overlap.

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

Gibson Assembly requires adjacent fragments to share 20-40 bp overlapping sequences so the exonuclease can expose complementary single-stranded regions that anneal.

  • Design overlaps into PCR primers: each primer has a 3’ region (~20 bp) binding the template, plus a 5’ tail (~20+ bp) matching the adjacent fragment.
  • Verify fragment sizes on a gel before assembling. Wrong band size = failed reaction.
  • Purify fragments: PCR reactions contain enzymes, buffers, and dNTPs that interfere with Gibson. Use column purification to get clean DNA first.
  • Check for unintended internal homology that could cause fragments to misassemble at the wrong junctions.
  • Note: restriction digest fragments don’t inherently have overlaps, so they’d need overlaps added via PCR before Gibson, or should be assembled using ligation instead.

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

Heat shock: Cells are made competent with CaCl₂ on ice (Ca²⁺ neutralizes negative charges on DNA and membrane). Then a sudden 42°C heat shock for ~30 seconds creates transient pores in the membrane, allowing DNA to enter. Cells go back on ice to reseal, then recover in rich media (no antibiotics) for ~1 hour so they can start expressing the plasmid’s resistance gene.

Electroporation: An electrical pulse creates temporary membrane pores. More efficient than heat shock but needs specialized equipment.

Both methods are inefficient (most cells die or don’t take up the plasmid), but antibiotic selection solves this. The plasmid carries a resistance gene, and cells are plated on agar with that antibiotic. Only cells with the plasmid survive and form colonies.

6. Describe another assembly method in detail: BioBrick Assembly

BioBrick is the iGEM standard. Its power is universal part compatibility rather than chemical cleverness.

The standard: every BioBrick part has the same flanking restriction sites. Left end (prefix): EcoRI + NotI. Right end (suffix): SpeI + PstI. Every part in the iGEM registry uses this same structure.

Joining two parts: Cut Part A with EcoRI + SpeI. Cut Part B with XbaI + PstI (XbaI and SpeI produce compatible sticky ends). Ligate. The SpeI-XbaI junction forms an 8 bp “scar” that can’t be re-cut by either enzyme. The combined AB part still has the same prefix/suffix on its outer ends, so it can be joined to Part C using the exact same process. Infinitely chainable.

Advantages: any part works with any other part, thousands available in the iGEM registry, simple protocol, easy to teach.

Disadvantages: one part at a time (each addition = cut, ligate, transform, select, verify), the scar can disrupt reading frames, restriction sites can’t appear inside your parts. Gibson and Golden Gate are technically faster for multi-fragment work.

See hand-drawn diagram below showing BioBrick prefix/suffix structure and two-part joining.