Week 6 HW: Genetic Circuits Part I: Assembly Technologies

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

Phusion High-Fidelity PCR Master Mix with HF Buffer is a 2X master mix consisting of Phusion DNA Polymerase, deoxynucleotides and reaction buffer that has been optimized and includes MgCl2. All that is required is the addition of template, primers and water. Taking each component and explaining its purpose:

  • Phusion DNA Polymerase: Ensures high accuracy when copying the amilCP gene and surrounding regions.
  • dNTPs: Provide the raw materials for synthesizing new DNA strands, including the chromophore-coding mutations.
  • MgCl₂: Acts as a cofactor for the polymerase, ensuring efficient and accurate DNA replication. Affects how well primers bind, which is important since your primers contain intentional mismatches for mutagenesis.
  • Reaction Buffer (with KCl and Tris-HCl): KCl helps primers bind properly to the template DNA, ensuring efficient amplification of the two amplicons. Tris-HCl maintains a stable pH, keeping the polymerase active during the reaction. (DNA Polymerase works best within a specific pH range: 7.4 -8.3)
  • Stabilizers and Enhancers: Some enhancers may assist in dealing with GC-rich regions or secondary structures in the amilCP gene.

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

  • Primer Length:

    Longer primers (≥20 bases) have more hydrogen bonds with the template DNA, requiring a higher annealing temperature.

    Shorter primers (<15 bases) bind more weakly and need a lower annealing temperature.

  • Primer Sequence Composition (GC Content):

    Guanine (G) and Cytosine (C) pairs form three hydrogen bonds, making them stronger than Adenine (A)–Thymine (T) pairs, which have only two hydrogen bonds.

    Higher GC content means higher Tₐ because more energy is needed to break the bonds.

    Lower GC content means lower Tₐ since the bonds are weaker.

  • Salt Concentration (Mg²⁺ in Buffer):

    Mg²⁺ stabilizes primer-template binding, making annealing more efficient.

    Higher Mg²⁺ → stabilizes base pairing → slightly higher Tₐ.

    Too much Mg²⁺ → nonspecific binding → more primer-dimer formation.

  • Primer Mismatches:

    If a primer has mismatches with the template, it binds less efficiently.

    This lowers Tₐ because weaker interactions make it easier for the primer to detach.

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 and restriction enzyme digest are two by which linear DNA fragments can be obtained, but they differ in mechanism and use.

PCR amplifies a specific DNA fragment using a thermostable DNA polymerase, primers, and repeated cycles of denaturation, annealing, and elongation. The process takes about 1–2 hours and produces large amounts of DNA, with the exact sequence defined by the primers. It is preferred when you want to amplify a specific region, introduce mutations, or when suitable restriction sites are not available.

Restriction enzyme digestion cuts DNA at specific sites using endonucleases, by incubation at an optimal temperature (usually37∘C). It does not amplify DNA, but generates fragments with cohesive or blunt ends. It is preferred for cloning, especially when you need compatible ends or are working with large DNA fragments.

In practice, the two methods are often combined to obtain fragments amplified and prepared for insertion into vectors.

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

Check for correct overhangs in PCR primers or restriction digest sites. Gibson Assembly requires overlapping DNA sequences at the ends of fragments. Each fragment must have 20-40 bp of sequence homology with the adjacent fragment.

  • Verify PCR fragment size on a gel and purify if necessary. PCR must accurately amplify the DNA regions needed for Gibson Assembly Run agarose gel electrophoresis to confirm correct fragment sizes..
  • Ensure complete restriction digestion of plasmid backbone. Use restriction enzymes that leave blunt or compatible sticky ends for seamless assembly. Run a control digestion and analyze by gel electrophoresis.
  • Purify DNA to remove contaminants. Purify DNA using a PCR cleanup kit or ethanol precipitation.

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

The plasmid DNA enters the E. coli cells during transformation by electroporation or thermic shock.

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

Golden Gate assembly is a very efficient cloning method, used to stick many DNA fragments in a single reaction. The method uses type IIS restriction enzymes and a ligase. Cloning is performed by pipetting in a single tube all plasmid donors, the recipient vector, a type IIS restriction enzyme and ligase, and incubating the mix in a thermal cycler. Type IIS enzymes reconize a specific DNA sequence, but cut outside of it, allowing for overhangs designed by the researcher. Fragments with complementary overhangs can line up in the correct order, and the ligase seals the bonds between them. Because the recognition sequence can be removed after assembly, the final product is usually “scarless,” meaning it leaves no additional marks at the junctions. This method is very useful for directional and modular assembly of multiple DNA fragments.

Golden Gate is best when you want standardized parts, directional multi-fragment assembly, and a scarless final construct using Type IIS sites.

Gibson is best when you want to join fragments based on longer homologous overlaps and do not want to rely on restriction sites at all.

So, Gibson is often easier for custom designs, while Golden Gate is often stronger for combinatorial and library-style cloning.

Assignment: Asimov Kernel

Click here to view my Asimov Kernel Notebook

Summary of Completed Work:

  1. Repressilator Recreation: Successfully recreated the classic 3-gene negative feedback oscillator loop and verified the periodic protein concentration waves using the dynamic simulator.
  2. Circuit 1 - Simple Toggle Switch: Documented a bistable switch mechanism demonstrating mutually exclusive states based on reciprocal repression.
  3. Circuit 2 - Delayed GFP Expression: Modeled a transcriptional activation cascade that produces a distinct time-delay in fluorescence.
  4. Circuit 3 - Auto-Repression GFP Circuit: Simulated a negative feedback loop that quickly stabilizes protein concentrations into a reliable plateau.