Week 6 HW: Genetic Circuits part I

Assignment: DNA Assembly

Answer these questions about the protocol in this week’s lab:

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

R/The Phusion HF PCR Master Mix contains several key components. The Phusion DNA Polymerase is a high-fidelity polymerase with 3’→5’ exonuclease (proofreading) activity, which significantly reduces errors during amplification. dNTPs (dATP, dTTP, dCTP, dGTP) serve as the building blocks for the new DNA strand. The HF Buffer stabilizes pH and ionic conditions optimal for standard templates, while the GC Buffer is used for GC-rich sequences. MgCl₂ acts as an essential cofactor for polymerase activity. Additional stabilizers and additives improve enzyme stability and overall reaction efficiency.

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

R/Several factors influence the annealing temperature of primers during PCR. The GC content of the primer is a major factor, since G≡C base pairs have three hydrogen bonds (stronger than A=T), so higher GC content raises the melting temperature (Tm). Primer length also matters — longer primers have a higher Tm. The standard rule is to set the annealing temperature approximately 5°C below the lowest Tm of the two primers. Salt concentration in the reaction buffer stabilizes the DNA duplex and raises Tm. Mismatches between the primer and template lower the Tm. Finally, design tools such as NEB’s Tm Calculator or Primer3 account for all these variables and help determine the optimal annealing temperature.

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.

R/Both PCR and restriction enzyme (RE) digestion generate linear DNA fragments, but they differ significantly in mechanism and application. PCR amplifies a target sequence through repeated cycles of denaturation, annealing, and extension in a thermocycler, using two primers that define the fragment’s ends. Restriction enzyme digestion, by contrast, incubates DNA with a sequence-specific enzyme (typically at 37°C) that cuts at defined recognition sites. A key advantage of PCR is flexibility: the primers can be designed to add any desired overhang sequences to the ends of the fragment, which is especially useful for Gibson Assembly. Restriction digestion is limited to cutting wherever recognition sites naturally exist in the sequence. PCR is preferable when you need to amplify a fragment from a template and simultaneously engineer specific end sequences. RE digestion is preferable when the fragment already exists in a plasmid with convenient restriction sites, or when you want to avoid the small but real risk of polymerase-introduced mutations. PCR requires minimal starting material, while RE digestion requires sufficient amounts of purified DNA containing the target sequence.

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

R/Gibson Assembly requires that adjacent fragments share homologous overlaps of 15–40 bp at their ends. For PCR fragments, primers must be designed with 5’ extensions that are homologous to the adjacent fragment or vector. These extensions do not anneal to the template during PCR but are incorporated into the final product, creating the necessary overlaps. For restriction enzyme-digested fragments, the ends must be verified to share sequence with the neighboring fragment, or overlaps can be added through a subsequent PCR step. Before performing the experiment, an in silico simulation using tools like Benchling or SnapGene should be done to confirm that all overlaps are correct and in the right orientation. Gel electrophoresis of all fragments prior to assembly confirms they are the correct size and free of contamination.

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

R/The most common method used in the lab is chemical transformation. First, cells are made competent by treatment with ice-cold CaCl₂, which destabilizes the cell membrane and allows it to interact with DNA. The plasmid is then mixed with the competent cells on ice. A brief heat shock at 42°C for approximately 30–45 seconds causes a sudden expansion of the membrane, creating transient pores through which the plasmid DNA can enter the cell. The exact molecular mechanism is not fully understood, but the CaCl₂ treatment is thought to neutralize the negative charges on both the DNA and the membrane, facilitating their interaction. An alternative method, electroporation, uses a high-voltage electrical pulse to physically create pores in the membrane, allowing DNA entry. After transformation, cells are allowed to recover in rich media before being plated on selective antibiotic plates.

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).

R/Golden Gate Assembly is a DNA assembly method that uses Type IIS restriction enzymes, such as BsaI, which cut downstream of their recognition sequence rather than within it. This property allows researchers to design any desired 4-nucleotide overhang at the cut site, independent of the enzyme’s recognition sequence. Each DNA fragment is designed so that the Type IIS recognition sites flank the insert and are oriented to cut outward, removing the recognition site from the final product. When the enzyme cuts, it generates unique 4-nt overhangs on each fragment that are complementary only to the correct neighboring fragment, ensuring directional and specific assembly. A DNA ligase (T4 DNA Ligase) then seals the fragments together. The entire reaction — digestion and ligation — can be performed simultaneously in a single tube by thermocycling between 37°C (digestion) and 16°C (ligation), allowing incorrect assemblies to be re-cut and re-ligated until the correct product accumulates. Because the recognition sites are eliminated from the final product, Golden Gate Assembly is scarless, and it can assemble up to 20 or more fragments in a single reaction, making it highly efficient for building complex genetic constructs.

Model this assembly method with Benchling or Asimov Kernel!

Assignment: Asimov Kernel

Create a Repository for your work

Create a blank Notebook entry to document the homework and save it to that Repository

Explore the devices in the Bacterial Demos Repo to understand how the parts work together by running the Simulator on various examples, following the instructions for the simulator found in the “Info” panel (click the “i” icon on the right to open the Info panel)

Create a blank Construct and save it to your Repository

Recreate the Repressilator in that empty Construct by using parts from the Characterized Bacterial Parts repository

Search the parts using the Search function in the right menu

Drag and drop the parts into the Construct

Confirm it works as expected by running the Simulator (“play” button) and compare your results with the Repressilator Construct found in the Bacterial Demos repository

Document all of this work in your Notebook entry - you can copy the glyph image and the simulator graphs, and paste them into your Notebook

Build three of your own Constructs using the parts in the Characterized Bacterials Parts Repo

Explain in the Notebook Entry how you think each of the Constructs should function

Run the simulator and share your results in the Notebook Entry

If the results don’t match your expectations, speculate on why and see if you can adjust the simulator settings to get the expected outcome