Simulate Gibson Assembly®

How do I simulate Gibson Assembly in SnapGene?

Gibson Assembly® is a registered trademark of Synthetic Genomics, Inc. For background on Gibson Assembly® see https://en.wikipedia.org/wiki/Gibson_assembly.

To watch a video on Simulating Gibson Assembly® in SnapGene see – this link.

In this lesson we will simulate directional in-frame fusion of the SEC13 coding sequence (CDS) and mEGFP CDS into the Pichia pastoris expression vector pIB2.

Summary of steps in this lesson:

1. Define the insert point in the vector sequence between KpnI and SpeI sites.

2. Define the two insert fragments for assembly with the vector sequence.

3. Let SnapGene design primers for PCR amplification of the two inserts to allow Gibson Assembly of the vector and insert fragments.

4. Simulate a Gibson assembly reaction between the vector and insert fragments to generate a circular product.

Open the Vector Sequence

Open the vector sequence, in Map view or Sequence view, click on the first restriction site, then shift click on the second restriction site to select the region that will be replaced by the insert fragments.

Start the Gibson Assembly Tool

Click Actions → Gibson Assembly® → Insert Two Fragments.

When starting the Gibson Assembly tool, the DNA sequence selection in the foremost window will be automatically set as the vector region to be replaced by Gibson Assembly!

The Gibson Assembly tool opens at the "Vector" tab, with the option to "Linearize with restriction enzymes" automatically selected and the appropriate fragment for replacement selected.

Define the First Insert Fragment

Switch to the "Fragment 1" tab, set the "Source of Fragment 1" via the dropdown menu, or click Browse to locate the insert sequence file on your computer.

By default SnapGene will assume you will need to amplify the selection by PCR. The option to "Use as a template for PCR" will be automatically set.

In Map view, select the Feature defining the Fragment 1 insert, or switch to Sequence view and manually select the insert region.

In this example, we wish to generate an in-frame fusion between the SEC12 CDS and the mEGFP CDS.

Switch to Sequence view and select the precise sequence region to exclude the SEC13 stop codon.

Define the Second Insert Fragment

Switch to the "Fragment 2" tab, set the "Source of Fragment 2" via the dropdown menu, or click Browse to locate the insert sequence file on your computer.

By default SnapGene will assume you will need to amplify the selection by PCR. The option to "Use as a template for PCR" will be set.

In Map view, select the Feature defining the insert, or switch to Sequence view and manually select the insert region.

Design Primers to Amplify the Insert Fragments

Switch to the Product tab and click Choose Overlapping PCR Primers.

Set the desired Target Tm for the PCR primers.

Set the desired overlap length and the target Tm for the overlap regions.

Click Choose Primers to design PCR primers for amplification of the inserts.

Validate the Vector/Insert Fusion Points

In the Product tab, switch to Sequence view. Scroll and check the fusion boundaries between each fragment.  In this example we confirm that the SEC13 CDS is in-frame with the mEGFP CDS.

Name the New Primers and Create the Product Sequence

Switch to each Fragment tab in turn, set appropriate names for each new primer.

Set an appropriate name for the Product sequence, then click Assemble to create the Product sequence.

Click File → Save to save the Product sequence to an appropriate location on your computer.

View History

Switch to History view to see all steps simulated and sequences/primers created during the Gibson Assembly procedure.

Order the PCR primers

See Export Primers for details on how to order the new primer sequences.