Week 6 — PCR, Gibson Assembly & Genetic Circuits

Part A: Pre-Lab Protocol Questions

1. Phusion High-Fidelity PCR Master Mix

According to the NEB product page, the Phusion master mix is built around two key features of the Phusion polymerase itself:

  1. A Pyrococcus-like polymerase core with a 3’→5’ proofreading exonuclease — catches and corrects misincorporated bases in real time, giving ~50x lower error rate than Taq.
  2. An Sso7d processivity domain fused to the polymerase — a DNA-binding clamp that keeps the enzyme on the template, increasing speed and fidelity together.

The master mix also includes dNTPs, MgCl₂ (essential cofactor for polymerase activity), and a reaction buffer optimized for Phusion.

2. Factors That Determine Primer Annealing Temperature

The annealing temperature is set based on the primer’s melting temperature (Tm) — the temperature at which half the primer-template duplexes dissociate. Two main factors drive Tm:

  1. GC content — G-C base pairs form 3 hydrogen bonds vs. 2 for A-T pairs, so GC-rich primers require more energy to dissociate and have a higher Tm.
  2. Primer length — longer primers have more total bonds to the template, raising Tm.

Salt concentration also plays a minor role — higher salt stabilizes the duplex and raises Tm slightly.

In practice, Tm is calculated using the NEB Tm Calculator with Phusion selected as the polymerase. NEB recommends setting the annealing step at Tm or Tm +3°C for Phusion, which is higher than for Taq due to Phusion’s greater processivity. In a standard PCR cycle this sits between the denaturation step (94–96°C) and extension (72°C). The ideal annealing temperature in practice is 52–58°C — going above 65°C risks secondary annealing, where primers bind non-specifically to off-target sites.

3. PCR vs. Restriction Enzyme Digests

This is essentially the distinction between scarred and scarless cloning approaches.

Restriction enzyme digests cut at specific recognition sequences and create sticky-end overhangs. The recognition sequence remains in the final construct — leaving a scar at the junction. Simple and fast when compatible sites already exist on the insert and vector, but you’re constrained by where those sites naturally appear.

PCR lets you directly extract any region regardless of restriction sites, and primer tails can add any overhang, mutation, or homology region needed. This is what makes scarless assembly methods like Gibson possible — the overlap is designed directly into the primers, with no recognition sequence left behind.

Prefer RE digests when compatible sites already exist and the workflow needs to be simple. Prefer PCR when no convenient sites exist, when precise scarless junctions are needed, or when building multi-fragment assemblies.

4. Ensuring Sequences are Compatible for Gibson Cloning

Two things need to be in place before Gibson Assembly will work:

Overlaps — Gibson requires identical sequence at every junction between adjacent fragments. These overlaps don’t exist naturally, so they’re designed into the PCR primers: each primer has two regions — an 18–22 bp binding region that anneals to the template during PCR, followed by a 20–22 bp overhang matching the end of the adjacent fragment. The PCR product therefore comes out with the overlap already attached. The Gibson mix’s 5’ exonuclease chews back those ends to expose single-stranded overhangs that anneal, get filled, and get ligated into a seamless join. Overlaps should be verified in Benchling or SnapGene before ordering — align fragments and confirm homology at each junction, avoiding repetitive sequences or secondary structures in the overlap region.

Codon optimization — when codon-optimizing the insert sequence, the algorithm can freely swap synonymous codons and may accidentally introduce restriction enzyme recognition sequences inside the coding sequence. While this doesn’t affect Gibson directly, it can cause problems if restriction enzymes are used elsewhere in the workflow. Tools like IDT and Twist allow you to blacklist specific restriction sites during optimization, which is worth doing as a precaution before ordering.

5. How Does Plasmid DNA Enter E. coli During Transformation?

In the lab we used chemical transformation (heat shock). The process works in three stages:

  1. Ice — competent cells (prepared with CaCl₂, which partially destabilises the membrane) are mixed with plasmid DNA and kept on ice. The cold temperature keeps everything stable and allows the negatively charged DNA to associate with the outer membrane surface.
  2. Heat bath (42°C, ~45 sec) — the brief temperature spike creates transient pores in the membrane, through which the DNA enters the cell.
  3. Back to ice — immediately halts the heat shock and stabilises the cells before recovery in SOC medium at 37°C. Cells are then pelleted by centrifuge and plated on selective agar.

An alternative method is electroporation, where a brief high-voltage pulse creates temporary membrane pores. It offers higher transformation efficiency and works well for large plasmids or low DNA concentrations, but requires specialised cuvettes and equipment.

6. Golden Gate Assembly

Two useful analogies help distinguish Gibson and Golden Gate:

Gibson Assembly is like a zipper. PCR primers add long overlapping sequences (~25 bp) to each fragment end. An exonuclease then rips back one side, exposing a single-stranded tail — which finds its complement on the adjacent fragment and zips together. It works because the sequences are identical mirrors of each other.

Golden Gate is like melodies blending seamlessly. Rather than overlapping, each fragment ends with a short 4-note motif (a 4-nt sticky end) that hands off directly into the opening motif of the next fragment. One musical phrase resolves into the next — no repeated section, no scar. The BsaI recognition site is like an intro riff that gets cut away before the song properly starts: it does its job generating the sticky end, then disappears from the final construct entirely.

Because each 4-nt overhang is unique, every fragment knows exactly which neighbour to find — making it possible to assemble many fragments simultaneously in a single pot reaction, with high efficiency and no scars at junctions.

Part B: Asimov Kernel

Note: Part B uses the Asimov Kernel platform. As a Global Committed Listener I did not have access to Asimov during the course — this section is left incomplete.

The Repressilator

The Repressilator is a synthetic genetic oscillator built from three transcriptional repressors wired in a loop: LacI represses tetR, TetR represses cI, and CI represses lacI. This negative feedback loop creates sustained oscillations in protein levels.

Repressilator construct — in progress.

Custom Constructs

Construct 1

In progress.

Construct 2

In progress.

Construct 3

In progress.