Week 6: Genetic Circuits Part I - Assembly Technologies

Molecular Biology: PCR, Cloning & Transformation

1. Phusion High-Fidelity PCR Master Mix Components

• Phusion Hot Start II DNA Polymerase — synthesizes new DNA strands; has 3’→5’ exonuclease (proofreading) activity to correct misincorporated bases, giving very high fidelity.
• dNTPs (dATP, dCTP, dGTP, dTTP) — deoxynucleotide triphosphates; the building blocks incorporated into the growing DNA strand.
• MgCl₂ (magnesium chloride) — essential cofactor; Mg²⁺ ions stabilize the enzyme-DNA-dNTP complex and are required for catalytic activity.
• Optimized reaction buffer — maintains correct pH and ionic environment for efficient polymerase activity and primer annealing.
• Hot-start antibody/aptamer — inhibits polymerase at room temperature to prevent non-specific amplification; releases the enzyme once the initial high-temperature denaturation step is reached.

2. Factors Determining Primer Annealing Temperature

• GC content — G-C pairs have 3 hydrogen bonds vs. 2 for A-T; higher GC → higher Tm → higher annealing temperature.
• Primer length — longer primers have higher Tm due to more base-pair contributions to stability.
• Self-complementarity — hairpins or primer dimers reduce effective annealing temperature.
• Salt/ion concentration — higher Mg²⁺ or monovalent cations stabilize the duplex, raising Tm.
• Additives (formamide, DMSO) — destabilize base pairing, lowering effective Tm; useful for GC-rich regions.
• Mismatches — imperfect complementarity (e.g., mutagenic primers) requires lower annealing temperature.

💡 Rule of thumb: set annealing temperature ~5°C below the calculated Tm of the primer pair.

3. PCR vs. Restriction Enzyme Digest

When to prefer PCR

• No convenient restriction sites flanking the sequence of interest
• Need to add sequences (e.g., overhangs for Gibson assembly) to fragment ends
• Amplifying from genomic DNA or low-abundance template

When to prefer Restriction Digest

• Working with a plasmid with known, convenient restriction sites
• Need defined sticky ends for directional ligation cloning
• Fidelity is paramount and PCR errors are a concern
• Linearizing a vector backbone

4. Ensuring DNA is Appropriate for Gibson Cloning

Gibson assembly requires 20–40 bp homologous overlaps between adjacent fragments.

For PCR Fragments

• Design primers so the 5’ end carries the overlap sequence homologous to the neighboring fragment; the 3’ portion anneals to the template.
• After PCR, the product carries the designed overlap at each end.
• Verify primer design computationally before ordering.

For Restriction-Digested Fragments

• Confirm that after digestion, the ends of each fragment are adjacent to and share sequence with the neighboring fragment.
• May require blunting or fill-in steps.

In Both Cases

• ✅ Run on an agarose gel to confirm correct fragment size
• ✅ Sanger sequence PCR products to rule out polymerase errors
• ✅ Confirm overlaps are unique (not repetitive)
• ✅ Check overlaps have no secondary structure that could interfere with the exonuclease in the Gibson mix

5. How Plasmid DNA Enters E. coli During Transformation

Heat-Shock Transformation (Chemical Competence)

1. Competence preparation — cells are treated with cold CaCl₂; Ca²⁺ neutralizes negative charges on DNA and the outer membrane, reducing electrostatic repulsion.
2. DNA binding — plasmid associates with the outer surface of the cell.
3. Heat shock — brief shift to 42°C (~30–45 sec) then back to ice; creates transient membrane instabilities/pores that drive DNA into the cell.
4. Recovery — cells incubate in SOC media at 37°C; repair membranes and begin expressing antibiotic resistance from the plasmid.
5. Selection — plated on antibiotic agar; only transformants survive.

Electroporation (Alternative)

• A high-voltage electric pulse creates transient pores in the membrane through which DNA enters.
• More efficient than heat shock; requires electrocompetent cells and specialized equipment.

6. Golden Gate Assembly

Overview

Golden Gate Assembly is a scarless, one-pot DNA assembly method using Type IIS restriction enzymes (e.g., BsaI, Esp3I) and DNA ligase.

How It Works

• Type IIS enzymes bind a defined recognition motif but cut outside of it — BsaI cuts 1 nt downstream on one strand and 4 nt on the other, generating a custom 4-nt 5’ overhang.
• Primers are designed so that after cutting, the recognition site is removed — leaving only the desired junction with no scar.
• Each junction has a unique 4-nt overhang, enforcing a single correct assembly order.
• The reaction thermally cycles between ligation (~16°C) and digestion (~37°C), progressively driving assembly toward the complete, ligated product — which no longer contains BsaI sites and cannot be re-cut.

Advantages

• Assemble 5–10+ fragments simultaneously in ~1 hour
• Junctions are seamless (no extra bases left behind)
• Directionality is enforced by unique overhangs
• Ideal for combinatorial library construction

Diagram

BEFORE DIGESTION:
  Vector:  5'--[BsaI]--ATCG|--backbone--3'
  Insert:  5'--insert--ATCG--[BsaI]--3'
                    ↓ BsaI cuts at | 

AFTER DIGESTION:
  Vector overhang:  5'--ATCG (4-nt overhang)
  Insert overhang:       ATCG--5' (complementary)

AFTER LIGATION:
  5'--backbone--ATCG--insert--ATCG--backbone--3'
      (BsaI recognition site is gone → no re-cutting → stable product)

MULTI-FRAGMENT ASSEMBLY:
  [Vector] ──AAAA→  ←AAAA──[Frag 1]──TTGC→  ←TTGC──[Frag 2]──CCGT→  ←CCGT──[Vector]
            unique        unique            unique
            overhangs enforce order → only one correct assembly possible

When to Use Golden Gate

• Assembling multiple fragments in a defined order
• Scarless junctions are critical (e.g., within coding sequences)
• Building modular or combinatorial construct libraries