Week 6 HW: Genetic Circuits Part I: Assembly Technologies

DNA Assembly

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

What you add yourself (not in the master mix) Forward primer + Reverse primer — define what region to amplify Template DNA — the sequence to be copied Nuclease-free water — to bring the reaction to final volume

The star component is Phusion polymerase itself — it has a fused processivity domain that makes it faster than standard Taq, and crucially it has 3′→5′ proofreading (exonuclease) activity, giving it an error rate ~50× lower than Taq. This is why it is preferred when accuracy matters, such as before Gibson assembly or cloning.

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

The four main factors are GC content, primer length, the calculated melting temperature (Tm), and the buffer’s salt concentration. With Phusion specifically, NEB recommends using their online Tm calculator rather than generic formulas, because the enzyme’s processivity domain affects optimal conditions. A practical starting point is to set annealing temperature to the Tm of your lower-Tm primer minus 5°C, then optimise from there.

Consequences of wrong annealing temperature Ta too LOW → primers bind non-specifically → multiple bands, wrong products Ta too HIGH → primers cannot bind stably → no product or very low yield Rule of thumb for Phusion: use the NEB Tm Calculator and set Ta = Tm of lower primer − 5°C Gradient PCR can be used to empirically optimise Ta when uncertain

  1. 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.

Both methods produce linear DNA fragments, but through fundamentally different mechanisms. PCR synthesises new copies of a specific region exponentially from a template, using primers to define the exact endpoints — which means you can engineer the ends freely by adding sequences to your primers. A restriction enzyme digest cuts existing DNA at fixed recognition sequences, producing ends that are dictated entirely by where those sequences happen to sit in the molecule. In terms of error risk, PCR introduces the possibility of polymerase errors (minimised by Phusion’s proofreading), while RE digests are faithful to the original sequence but can only cut where recognition sites exist. If those sites don’t exist in your insert, or if a cut site would fall in an undesirable location, PCR is the only option. Conversely, if you already have a plasmid carrying your insert flanked by known restriction sites, a digest is faster and simpler than designing and running PCR.

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

Gibson assembly works by joining fragments that share 15–40 bp of identical sequence at their junctions. The exonuclease in the Gibson mix chews back 5′ ends to create single-stranded overhangs, which then anneal and are ligated. For your fragments to be compatible, you must ensure overlapping homology at every junction.

For PCR fragments, the key step is primer design — your forward primer for each insert should carry a 5′ tail of 20–40 bp that matches the end of the upstream fragment (or linearised vector). Tools like Benchling let you design these overlaps visually and verify them in silico before ordering primers. For RE-digested fragments, you need to confirm that the sticky or blunt ends of adjacent fragments are complementary and that no sequence is lost or mismatched at the junction. After generating your fragments by either method, always run a gel to confirm band size, then column-purify or gel-extract to remove primers, enzymes, and salts that would interfere with the Gibson reaction.

Key things that can go wrong Overlaps too short (<15 bp) → poor annealing, assembly fails Overlaps contain repeats or secondary structure → mis-assembly or rearrangements Impure fragments (primers still present) → compete with correct annealing

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

The most common lab method is heat-shock transformation of chemically competent cells. Cells are treated with divalent cations (typically CaCl₂) during preparation, which partially neutralises the negative charge on both the cell membrane and the DNA, reducing electrostatic repulsion. When the cold cell-DNA mixture is briefly shifted to 42°C, transient pores open in the membrane through which DNA can enter. The cells are then recovered in rich medium at 37°C (called the recovery or outgrowth step) to allow antibiotic resistance genes on the plasmid to be expressed before plating on selective media.

What happens at the molecular level CaCl₂ treatment: Ca²⁺ ions coat DNA and neutralise the membrane’s negative charge Ice incubation: DNA-cell complexes form and associate with the outer membrane Heat shock: membrane fluidity spike creates transient pores → DNA enters cytoplasm Return to ice: pores reseal, DNA is trapped inside Outgrowth: plasmid replicates, antibiotic resistance gene is expressed before selection

An alternative to chemical transformation is electroporation, where a brief electrical pulse physically creates pores in the membrane to allow DNA entry. Electroporation generally gives 10–100× higher efficiency and is preferred for large plasmids or when transformation efficiency is critical. However, it requires electrocompetent cells (washed to remove salts) and specialised equipment.

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

Golden Gate Assembly is an elegant cloning strategy that uses Type IIS restriction enzymes — enzymes that cut outside their recognition sequence to generate custom 4-bp overhangs. Because the enzyme cuts away from its own recognition site, the recognition sequence is eliminated from the final product, leaving only the overhangs you designed. This enables scarless, seamless assembly.

Golden Gate uses a Type IIS enzyme (most commonly BsaI or BsmBI) whose recognition sequence and cut site are spatially separated. Each DNA part is flanked by Type IIS recognition sequences oriented so that cutting releases the recognition site and leaves behind a unique 4-bp overhang. Because every junction has a distinct 4-bp overhang, the parts can only assemble in one correct order — the overhangs provide directionality and specificity simultaneously. The restriction enzyme and T4 DNA ligase are added to the same tube and thermocycled together: digestion and ligation alternate in repeated cycles, so misligated or incorrect assemblies are continuously re-cut and corrected while the correct assembly accumulates. The final assembled product lacks all Type IIS sites, so it can never be re-cut by the enzyme in the reaction. This makes Golden Gate highly efficient even for assemblies of 5–10 fragments in a single reaction, which makes it faster and more scalable than sequential cloning strategies.

ii. Model this assembly method with Benchling or Asimov Kernel!

  1. Go to benchling.com and create a free account (or log into your course account)
  2. Create a new DNA sequence — paste in your vector sequence
  3. Add annotations for each BsaI recognition site (GGTCTC for BsaI) using the annotation tool
  4. Create separate sequences for each insert part, each flanked by BsaI sites with the correct 4-bp overhangs
  5. Use the Assembly Wizard (under Cloning → Assembly) — select “Golden Gate” as the assembly type, add your parts in order, and Benchling will simulate the digestion and show you the predicted assembled construct
  6. Verify that the junctions are correct and the reading frame is maintained across each junction

As a comparison to Gibson: Golden Gate is preferable when you are assembling many parts (5+) in a defined order and want precise scarless junctions, while Gibson is simpler to set up for 2–3 fragments and does not require specific restriction sites to be absent from your inserts. Golden Gate requires that no internal BsaI sites exist within any of your parts (you may need to silently mutate them out), whereas Gibson has no such constraint.