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.
📷 Diagram credit: SnapGene Gibson Guide

The enzyme mix includes three key components:

5′ Exonuclease: Creates single-stranded 3′ overhangs by chewing back the 5′ ends.
DNA Polymerase: Fills in gaps after annealing.
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, we'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

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

🎥 Watch: Design of the Gibson Assembly plan¶

Step 1: Define Your Product¶

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

Open the pET28a vector sequence and convert it to UPPERCASE.
Open the insulin cDNA sequence and convert it to lowercase.
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.

🔗 Downloads:

Step 2: Design Oligos¶

After modeling your final plasmid with insert and vector joined, identify the junctions where fragments meet.

For each junction:

Select ~20 bp from the end of one fragment
Select ~20 bp from the start of the next fragment
Concatenate these to form a 40 bp primer

Use this 40 bp sequence directly as the forward oligo.
For the opposite junction, take the corresponding 40 bp and reverse complement it to create the reverse oligo.

When choosing the ~20, follow the same general approach for finding annealing sequences between 18 and 25 bp long, balanced base content, etc. as done previously in the Basic Cloning tutorial.

Step 3: Write the Construction File¶

Here's a complete construction file representing this cloning plan:

View full Construction File (CF) (click to expand)

PCR for_ins rev_ins insulin_cdna    ins_pcr 
PCR for_pet rev_pet pET28a  pet_pcr 
Gibson  ins_pcr pet_pcr     gibs    
Transform   gibs    Mach1   Kan 37  pET-INS

oligo   for_ins CTTTAAGAAGGAGATATACCATGGCCCTGTGGATGCGCCTC           
oligo   rev_ins GTGGTGGTGGTGGTGCTCGAGctagttgcagtagttctccag          
oligo   for_pet ctggagaactactgcaactagCTCGAGCACCACCACCACCAC          
oligo   rev_pet GAGGCGCATCCACAGGGCCATGGTATATCTCCTTCTTAAAG       
dsdna   insulin_cdna    agccctccaggacaggctgcatcagaagaggccatcaagcagatcactgtccttctgccatggccctgtggatgcgcctcctgcccctgctggcgctgctggccctctggggacctgacccagccgcagcctttgtgaaccaacacctgtgcggctcacacctggtggaagctctctacctagtgtgcggggaacgaggcttcttctacacacccaagacccgccgggaggcagaggacctgcaggtggggcaggtggagctgggcgggggccctggtgcaggcagcctgcagcccttggccctggaggggtccctgcagaagcgtggcattgtggaacaatgctgtaccagcatctgctccctctaccagctggagaactactgcaactagacgcagcccgcaggcagccccccacccgccgcctcctgcaccgagagagatggaataaagcccttgaaccaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
plasmid pET28a  AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCG

Step 4: Simulate the Gibson Assembly¶

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

Predict the PCR products using your designed primers anneal as expected
Identify the overlapping sequences at the ends of each fragment.
For each overlap, delete one copy so the fragments can join seamlessly.
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:
🔗 Use C6 simulation tools Copy the full text above including the sequences and paste it into the simulation tool.

These tools let you verify that your oligos will function 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.
- The atg of your CDS should overlap the NcoI site as 'CCatgg'
- Paste the CDS in lowercase, and keep the plasmid sequence in UPPERCASE.

2. Identify each junction¶

For each junction:
- Forward oligo = 20 bp before junction + 20 bp after junction
- Reverse oligo = reverse complement of forward oligo

3. 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¶

🧪 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.