Week 6 HW: Genetic Circuits Part I: Assembly Technologies

Q1 What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

  • Phusion DNA Polymerase which synthesises new DNA strands by adding nucleotides using the template during replication. It ensures high precision copying, editing and proofreading.
  • Deoxynucleotides - dNTP consists of a deoxyribose sugar, a nitrogenous base (A, T, C, or G), and three phosphate groups. The DNA polymerase adds them to a growing DNA chain to form the new complementary strand.
  • Reaction Buffer - creates a suitable chemical environment for the Phusion High-Fidelity DNA Polymerase for DNA synthesis. Regulates a stable pH, provides magnesium ions as a cofactor for catalytic activity, and contains important components to increase specificity, yield and high-fidelity performance.
  • Magnesium Chloride - DNA polymerase which synthesises DNA, requires Mg2+ for DNA synthesis, as it is required for enzyme Catalytic Activity, in the active site to catalyze the formation of phosphodiester bonds between the 3′-OH of a primer and the phosphate group of a nucleotide.
  • DMSO to increase G/C rich targets - additive which boosts denaturation of difficult or GC-rich targets.

Q2 What are some factors that determine primer annealing temperature during PCR?

  • G/C content of the primer sequence - higher GC content will have a higher melting temperature as G/C base pairs have three hydrogen bonds compared to two in A/T base pairings.
  • Longer primer sequences require a higher melting temperature, which need a higher primer annealing temperature.
  • Cations (like Na+) and magnesium ion concentration can cause the melting temperature to increase.
  • Mutations in the target sequence, and base pair mismatches between the primer and the target can reduce the stability and the annealing temperature
  • Denaturants which reduce stability of primer-template duplexes and reduce primer melting temperature

Q3 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 should be picked if:

  • There is less DNA starting material
  • No restriction enzyme cutting sites which are suitable for cutting near gene of interest
  • Want to introduce a selective mutation
  • Want to add sequences at the end (like in Gibson’s Assembly)

Restriction enzymes should be picked if:

  • Cutting a circular DNA plasmid into a linear molecule to allow for the insertion of a DNA insert, for cloning and ensuring 100% backbone sequence correctness
  • Checking if the plasmid has the right DNA insert
  • DNA is in high concentration and we have the correct sites for RE cutting

Q4 How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

  • High GC content in the overlaps to promote stability
  • Run PCR on gel to ensure correct sizes and no contaminants - ensure purification of the DNA from the gel to remove template DNA and enzymes
  • The fragments need to share 20–40 bp of homologous sequence at the ends of the insert(s) and linearised vector. Primers need to be designed with tails that match the adjacent fragment or vector.
  • Full digested vector needs to be linearised
  • Use High-Fidelity Polymerase

Q5 How does the plasmid DNA enter the E. coli cells during transformation?

For DNA entry to occur, cells must first be made competent.

This is achieved through two main methods:

  • Chemical transformation (heat shock): Cells are treated with calcium chloride to allow DNA binding to the cell surface. Quickly shifting the cells from an ice bath to a 42°C water bath creates a pressure difference that allows the DNA to enter the cell.
  • Electroporation: A short, high-voltage electric pulse is applied to the cell suspension, which physically creates temporary pores in the cell membrane through which the plasmid DNA can pass.

Q6 Describe another assembly method in detail (such as Golden Gate Assembly) Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online). Model this assembly method with Benchling or Asimov Kernel!

In Golden Gate cloning, circular DNA in plasmids or linear PCR products is mixed in a tube with the Type IIS restriction enzyme (which cuts outside of the recognition sites, producing specific 4-base pair overhangs which aren’t part of the final, seamless construct, as the sites are removed, the final construct is stable and will not be cut again, which allows digestion and ligation of DNA for creating genetic circuits without the purification step required), and DNA ligase enzyme to repair and join the DNA, and the tube is put in the PCR machine. This is then transformed into E coli and selected and propagated on the agar plate (Bird et al., 2022).

Diagram showcasing Golden Gate assembly.

       [Plasmid/PCR DNA 1]   [Plasmid/PCR DNA 2]   [Plasmid/PCR DNA 3]
        /           \     /           \     /           \
   --[BsaI]-- ---[BsaI]---[BsaI]--- ---[BsaI]--- ---[BsaI]--- 
      [Site1]     [Site2] [Site2]     [Site3] [Site3]     [Site4]

         |           |       |           |       |           |
         V           V       V           V       V           V
   [Overhang 1]----[Overhang 2]----[Overhang 3]----[Overhang 4]
   (Four-letter overhangs made - a sticky edge generated by digestion)

                       + T4 DNA Ligase (for joining DNA)
                       + Destination Vector (For transformation into *E Coli*)
                       + Type IIS Enzyme 
                       + 37°C/16°C Cycling (if the pieces are joined together incorrectly (with the Restriction enzyme recognition sites still attached), the scissors can cut them apart again. If they stick correctly and the RE recognition sites are removed, the scissors can't recognise them anymore.)
                               |
                               V
           [Final Assembled Product]
      [Vector]-<1>---[Fragment 1]---<2>---[Fragment 2]---<3>---[Fragment 3]---<4>
      (Stable, restriction sites removed, precise ordering)
      
(Bird et al., 2022)

Key 

BsaI - RESTRICTION ENZYMES

Overhang - single-stranded DNA that stick out from the end of a double-stranded molecule.

Q6 (Part 2) To model Golden Gate Assembly in Benchling or Asimov Kernel, I start by digitizing my DNA fragments and cleaning them to remove any internal BsaI sites that might cause unwanted cutting. I then use the software’s Type IIS assembly tool to define my 4-base-pair overhangs, ensuring each piece slots into the next like a unique puzzle piece. I run a dry lab simulation to strip away the recognition sites and visualize the final, scarless plasmid map. This digital step is how I catch sequence errors and ensure my reading frames stay intact before I ever pick up a pipette.

This is the modelling in Benchling: https://benchling.com/s/seq-q2XW9o9mcjLBiH2V3lSA?m=slm-ex0hzqOg6DbZToMfb5Hi

The insert is GFP and the vector plasmid is pGDR.

Screenshot 2026-03-26 at 09.23.22.png Screenshot 2026-03-26 at 09.23.22.png

References

Bird, J. E., Marles-Wright, J., & Giachino, A. (2022). A user’s guide to golden gate cloning methods and standards. ACS Synthetic Biology, 11(11), 3551–3563. https://doi.org/10.1021/acssynbio.2c00355

New England Biolabs. (n.d.). Phusion High-Fidelity PCR Master Mix with HF Buffer. https://www.neb.com/en-gb/products/m0531-phusion-high-fidelity-pcr-master-mix-with-hf-buffer