Cloning for Synthetic Biology 2

Steps of Gibson Assembly


Core Concept

Fragments are designed with 20–40 bp overlapping homologous ends.

Reaction Conditions

  • Single tube

  • ~50°C (isothermal)

  • 3 enzymes

Step-by-Step Mechanism

  1. Design fragments with overlap regions

    • Added using tailed PCR primers (Fig. 7.07A)

  2. Add enzyme mix

    • 5′ exonuclease

    • DNA polymerase

    • DNA ligase

  3. 5′ Exonuclease activity

    • Chews back 5′ ends

    • Creates 3′ single-stranded overhangs

  4. Annealing

    • Complementary overlap regions anneal spontaneously

  5. DNA polymerase

    • Fills in single-stranded gaps

  6. DNA ligase

    • Seals phosphodiester backbone

Result

  • Seamless construct

  • No restriction sites left behind

  • Can assemble multiple fragments simultaneously

2⃣ Steps of Gateway Cloning

(See Slides pp. 8–12; Fig. 7.08–7.10)

13-Cloning for synthetic biolog…

Gateway cloning is based on λ phage site-specific recombination.

Key DNA Sites

  • attB

  • attP

  • attL

  • attR

Two Main Reactions

🔹 Step 1: BP Reaction (Entry Clone Creation)

  • Insert with attB sites

  • Donor vector with attP sites

  • Uses Int (integrase)

Result:

  • Insert becomes flanked by attL sites

  • Creates Entry clone

  • ccdB gene replaced (toxic gene removed)

🔹 Step 2: LR Reaction (Expression Clone Creation)

  • Entry clone (attL)

  • Destination vector (attR)

  • Uses Int + Xis (excisionase)

Result:

  • Insert transferred into destination vector

  • attL + attR → attB

  • ccdB replaced by insert

  • Creates expression clone

3⃣ Advantages & Disadvantages

🔬 Gibson Assembly

Advantages

13-Cloning for synthetic biolog…

  • Multi-fragment assembly

  • No restriction site constraints

  • Seamless (no extra bases)

  • Flexible design

  • Fast (single reaction)

Disadvantages

  • PCR-dependent

  • Requires careful primer design

  • More complex primer construction

🧬 Gateway Cloning

Advantages

13-Cloning for synthetic biolog…

  • High efficiency

  • Directional

  • Excellent for high-throughput

  • Easy shuttling into many vectors

  • Modular system

Disadvantages

  • Expensive

  • Leaves att site “scar” sequences

  • Limited to Gateway-compatible vectors

  • Less customization freedom

4⃣ Comparison of Cloning Methods

(Slide Comparison Table, p.16)

13-Cloning for synthetic biolog…

Feature

RE Cloning

TA

TOPO-TA

Gateway

Gibson

Mechanism

Restriction + ligase

A–T pairing

A–T + Topoisomerase

Site-specific recombination

Homology-based assembly

Restriction sites needed

Yes

No

No

No

No

Ligase required

Yes

Yes

No

No

Yes (in mix)

Directional

Yes (2 enzymes)

No

Usually no

Yes

Yes

Multi-fragment

Limited

No

No

Limited

Excellent

Scarless

No

Minimal

Minimal

No (att sites remain)

Yes

Cost

Low

Low

High

High

Moderate

Design complexity

Moderate

Low

Low

Moderate

High

5⃣ Insertional Inactivation (General)

(Slides pp. 20–21; Fig. 7.12)

13-Cloning for synthetic biolog…

General Principle

Insertion of DNA into a marker gene disrupts its function.

Used to:

  • Identify recombinant plasmids

  • Distinguish vector-only vs vector+insert

Common disrupted genes:

  • lacZ

  • Antibiotic resistance genes

  • Fluorescent protein genes

6⃣ Insertional Inactivation of lacZ (Blue-White Screening)

(Slides pp. 22–24)

13-Cloning for synthetic biolog…

Vector Contains:

  • MCS inside lacZα gene

Two Outcomes After Transformation

🔵 No Insert (Empty Vector)

  • lacZ intact

  • β-galactosidase produced

  • Cleaves X-gal

  • Colonies turn blue

⚪ Insert Present

  • Insert disrupts lacZα

  • No functional β-galactosidase

  • X-gal not cleaved

  • Colonies remain white

Conclusion:

White colonies = likely recombinant plasmid