Week 6 HW: Genetic circuits part 1

DNA ASSEMBLY

Phusion High-Fidelity PCR Master Mix Components

Phusion HF PCR Master Mix is provided as a 2X stock that is diluted to a 1X concentration in the final reaction. While the sources do not list every chemical ingredient, they indicate its purpose is to amplify specific DNA sequences (such as the amilCP gene and the mUAV backbone) with high accuracy.

Standard high-fidelity master mixes like Phusion typically contain:

  1. Phusion DNA Polymerase: A highly accurate enzyme with proofreading activity to minimize mutations.
  2. dNTPs (Deoxynucleotide Triphosphates): The building blocks (A, T, C, G) used to synthesize the new DNA strand.
  3. Buffer: Maintains the optimal pH and ionic strength for the enzyme.
  4. $MgCl_2$: A necessary cofactor for the DNA polymerase to function.

Factors Determining Primer Annealing Temperature

The annealing temperature is critical for ensuring primers bind specifically to their targets. Key factors mentioned in the sources include:

  1. Melting Temperature ($T_m$): The annealing temperature is generally set 2–5°C below the lower $T_m$ of the primer pair.
  2. Binding Region Length: Primers should ideally have an 18–22 bp core binding region.
  3. GC Content: The binding region should ideally have a GC content of 40–60%.
  4. GC Clamp:Adding 1–2 G or C bases at the 3’ end promotes specific binding.
  5. Secondary Structures: Avoiding strong hairpins or dimers ensures the primer is available to bind the template.

PCR vs. Restriction Enzyme Digests

Both methods generate linear DNA fragments but differ significantly in application:

  1. Protocol: a)PCR uses thermal cycling (denaturation, annealing, extension) and DNA polymerase to synthesize millions of new copies of a specific region. b)A restriction enzyme digest uses specific proteins to cut existing DNA at recognized sequences.
  2. When to Use PCR: PCR is preferable when you need to introduce mutations (like the chromophore color changes), add specific overhangs for assembly, or amplify a gene from a template.
  3. When to Use Restriction Digests: Digests are often used to linearize a circular vector or as a diagnostic tool (e.g., using DpnI to remove methylated template DNA) to verify fragment sizes on a gel.

Ensuring Appropriateness for Gibson Cloning

To ensure your digested and PCR-amplified sequences are ready for Gibson Assembly, you must verify:

  1. Overlaps: Adjoining fragments must have 20–40 bp of sequence identity (overhangs) at their ends.
  2. Orientation: Confirm that all fragments have the correct 5′→3′ orientation with matching overlaps to form a circular plasmid.
  3. Purity: Use DpnI to digest the original methylated template plasmid. Then, perform DNA purification (silica adsorption) to remove enzymes and buffers.
  4. Verification: Run a diagnostic gel to confirm the fragments are the correct size and measure concentration (ideally >30 ng/µL).
  5. Molar Ratio:Use a 2:1 (insert:vector) molar ratio for the assembly reaction.

Plasmid Entry During Transformation

During transformation, the sources explain that the cell membrane is “opened up” by shocking the cells using either heat shock (abrupt temperature change) or electroporation (high voltage). This process creates pores in the bacterial cell wall. Once these pores are present, the plasmid DNA enters the E. coli cells by diffusion.

Alternative Assembly Method: Golden Gate Assembly

Golden Gate Assembly (GGA) is a molecular cloning method that allows for the simultaneous, seamless assembly of multiple DNA fragments. It utilizes Type IIS restriction enzymes, which cut DNA at a specific distance away from their non-palindromic recognition sites. This unique feature allows designers to create custom 4-base overhangs that do not contain the original restriction site. Because the restriction sites are placed such that they are “cut off” during the reaction, the assembled product cannot be re-digested, driving the reaction toward the final circular plasmid. This makes GGA a highly efficient “one-pot” reaction where digestion and ligation occur simultaneously. It is particularly popular for modular cloning systems (like MoClo) because of its high fidelity and speed.

Diagram

Input fragments

[BsaI]-[Fragment 1]-A │ ▼ A-[Fragment 2]-B │ ▼ B-[Vector]-[BsaI]

    ↓ Golden Gate Assembly

(BsaI digestion + DNA ligase)

Final plasmid

[Fragment 1]──A──[Fragment 2]──B──[Vector]

   Circular seamless plasmid

Modeling in Benchling

To model Golden Gate Assembly in Benchling, you would follow these steps:

  1. Import Sequences: Load your DNA parts and target backbone.
  2. Check for Internal Sites: Use the “Digest” tab to ensure your parts do not contain the Type IIS site (e.g., BsaI) internally.
  3. Open Assembly Wizard: Select “Create New” -> “DNA Assembly” -> “Golden Gate.”
  4. Define Parts: Select the backbone and the inserts. Benchling will automatically identify the 4-bp overhangs.
  5. Verify Overlaps: Ensure the overhangs are complementary (e.g., the 3’ end of Part 1 matches the 5’ end of Part 2).
  6. Finalize: Click “Assemble” to generate the new virtual plasmid and verify the sequence.

Asimov Kernel

Pending …