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Gibson Assembly

Why Gibson Assembly?

In the previous tutorial, you used restriction enzymes to insert the INS gene into a pET vector. This is a reliable method, but it has a key limitation: it depends on restriction sites being in the right places.

In synthetic biology, you often need to assemble multiple parts with precise control over sequence. This is where Gibson Assembly shines. It allows you to join DNA fragments without restriction sites—using only sequence overlaps.

🔬 How Gibson Assembly Works

Schematic diagram of Gibson Assembly, showing the three-enzyme process used to assemble DNA fragments with overlapping ends.
Figure: Gibson Assembly joins DNA fragments with overlapping ends in a seamless, scarless way. An exonuclease chews back 5′ ends, exposing complementary overhangs. These anneal, and a polymerase fills in gaps. A ligase seals the nicks, yielding a continuous double-stranded product.
đź“· Diagram credit: SnapGene Gibson Guide

The enzyme mix includes three key components:

  1. 5′ Exonuclease: Creates single-stranded 3′ overhangs by chewing back the 5′ ends.
  2. DNA Polymerase: Fills in gaps after annealing.
  3. DNA Ligase: Seals the nicks to produce a covalently closed DNA strand.

This method is scarless, seamless, and does not rely on restriction sites.

Overview of This Tutorial

In this exercise, you'll recreate the pET-INS plasmid—but using Gibson Assembly instead of restriction enzymes.

You’ll use PCR to create two fragments: - One containing the INS gene - One containing the pET28a backbone

You’ll design primers that add 20–30 bp overlaps, simulate the assembly, and validate the final construct.

Step 1: Define Your Product

Start by constructing a model of the final pET-INS plasmid:

  1. Open the pET28a vector sequence and convert it to UPPERCASE.
  2. Open the insulin cDNA sequence and convert it to lowercase.
  3. Paste the INS cds into the intended insertion site as done in the basic cloning tutorial.

This marks where the insert meets the vector, making it easier to plan overlaps.

đź”— Downloads:

Step 2: Annotate Overlaps and Annealing Regions

Once you’ve created a model of your final product, it’s time to define the key regions for Gibson primer design.

For each junction where two fragments will join:

  1. Choose the Overlap
    Pick 20–30 bp spanning the junction. This is the sequence that will guide fragment assembly via Gibson. Think of it like the annealing region of a primer—aim for balanced GC content, low secondary structure, and ideally end in a G or C. Create a feature in your sequence editor labeled overlap.

  2. Mark the Forward Anneal Region
    Starting at the junction and extending downstream, choose 20–30 bp that follow standard primer design rules. Label this forward anneal.

  3. Mark the Reverse Anneal Region
    Identify 20–30 bp upstream (5′) of the junction. This sequence lies on the coding strand, not its reverse complement. Label it reverse anneal.

Together, these features define the sequence pieces you'll need to construct your oligos in the next step.

Step 3: Design Oligos

Each oligo consists of two parts, built from the regions you labeled earlier:

  • The forward oligo is made from the full overlap followed by the forward anneal region.
  • The reverse oligo is made from the reverse anneal region followed by the overlap, then reverse complemented.

Here is one example solution:

PCR       oINS_F        oINS_R        insulin_cdna    ins_pcr
PCR       oVec_F        oVec_R        pET28a         vec_pcr
Gibson    ins_pcr       vec_pcr                      gib_pdt
Transform gib_pdt       Mach1         Amp            pET-INS

oligo     oINS_F        GATATACCatggccctgtggatgcgcctc
oligo     oINS_R        GTGGTGGTGCTCGAGctagttgcagtagttctccag
oligo     oVec_F        ctgcaactagCTCGAGCACCACCACCACCAC
oligo     oVec_R        catccacagggccatGGTATATCTCCTTCTTAAAG

Step 4: Simulate the Gibson Assembly

Start by simulating the assembly manually to understand what’s happening:

  1. Predict the PCR products using your designed primers, ensuring that the ends include the overlap, forward anneal, and reverse anneal regions as annotated.
  2. Identify the overlapping (overlap) sequences at the ends of each fragment.
  3. For each overlap, delete one copy so the fragments can join seamlessly.
  4. Join the trimmed fragments to form your final product.

Then try using simulation tools to automate the process:

  • ApE and Benchling allow you to simulate Gibson assemblies graphically.
  • You can also use the C6 Tools to simulate the full CF script:
    đź”— Use C6 simulation tools

These tools let you verify that your overlaps (overlap), forward anneal, and reverse anneal regions are correct and that the final sequence is as expected.

🎯 Try it yourself

In your quiz, you'll use the randomly selected gene from Bacillus atrophaeus UCMB-5137 as done before in the Basic Cloning tutorial. Also, you will again be cloning it into the NcoI and XhoI restriction sites generating the same product plasmid as before. However, this time, you will make that plasmid by Gibson assembly instead of traditional restriction enzymes.

In this challenge, you’ll use Gibson Assembly to insert your assigned gene into the pET28a plasmid between the NcoI and XhoI restriction sites.

Just like in the pET-INS example:

1. Start by modeling the final product

  • Start by recreating the pET28a + gene design you made in the basic cloning tutorial.
  • If you skipped that tutorial, no problem — follow the guidance here to recreate it:
    • Use the plasmid backbone from pET28a sequence file.
    • Retrieve your assigned gene sequence using the "Quiz Instructions" box above.
    • Insert the entire CDS into the region between the NcoI (CCATGG) and XhoI (CTCGAG) sites.
    • Paste the CDS in lowercase, and keep the plasmid sequence in UPPERCASE.

2. Annotate each junction

  • For both junctions between insert and vector:
    • Define an overlap region: 20–30 bp that spans the junction.
    • Choose a forward anneal region: 20–30 bp downstream of the junction.
    • Choose a reverse anneal region: 20–30 bp upstream of the junction (on the coding strand).

3. Design your oligos

  • Forward oligo = overlap + forward anneal
  • Reverse oligo = reverse anneal + overlap, then reverse complement

4. Build your Construction File (CF)

  • Your CF should include:
    • Two PCR steps
    • One Gibson step
    • One Transform step
    • Four oligo lines
  • You will name and assign components based on your own design choices

Tip: The sequences for your assigned gene (see name in the Quiz Instructions box above) and for pET28a are preloaded in the autograder—no need to define them in your CF.

A Note on Naming and Templates

When you're ready, paste your CF into the autograder below and simulate.

Gibson Quiz

Paste your Construction File (CF) below and click Simulate. You’ll see the resulting sequences, and if your design is valid, it will complete the quiz.


🧪 Try and Break It

Once your plan works, experiment!

  • Delete an overlap and see what happens.
  • Remove a PCR step.
  • Flip the insert direction.

Watch how the validation catches errors. This helps you understand how Gibson designs succeed—or fail.