Gibson Assembly Tutorial: Basic Gibson Assembly

We want to create a Decorin(DCN)-eGFP fusion gene using Gibson Assembly.

First, we open the Vector which already contains the eGFP. We want to insert DCN just before the start codon of the GFP, so highlight the GFP CDS annotation and zoom in to the 5' end. Turn Translation on if it's not already activated (on the right hand side of the Sequence Viewer click on 'Display' → 'Translation'). The Gibson assembly works by joining sequence extremities together, so first we have to provide this 'extremity' by linearizing our vector just before the ATG codon using either PCR or restriction digestion. As there is already a unique NcoI restriction site we can conveniently use this to digest our vector. To do this go to Cloning → Digest into Fragments. Ensure only NcoI is selected and click OK.





The resulting sequence now bears two 5' overhangs which will be digested by T5 exonuclease during the Gibson Assembly. Removal of this overhang will keep our protein in-frame, but will remove the start codon (ATG). In order to keep the ATG and not truncate the CDS in any way we can add it again to the 5' end of the vector. Turn on Allow Editing and add the missing 'ATG' bases manually as shown in the figure below. This manual editing will not affect the reaction, as manual insertions, deletions or replacements are considered during the Gibson operation and will be included in the primer extension. Save the changes (deactivate link to parent, we won't need this link). Scrolling to the very end of the Vector you will notice the other CATG NcoI overhang. Here we don't have to do anything as we're not interested in this sequence part as long as it doesn't block transcription.



We're finished with the vector, so let's head over to DCN. We want only the CDS to be inserted before GFP, without the stop-codon. Translation should still be turned on, select the CDS bases except for the last UAA (for example by clicking on the CDS annotation, then holding Shift+click just before the 'UAA'). We're going to extract this without the use of primers, they will be generated later by Geneious during the Gibson operation. Furthermore Geneious will automatically convert the Uracil RNA bases to Thymine, so we don't have to worry about that either. Click on Extract in the Sequence Viewer and chose an appropriate name, such as "DCN CDS".

Now select both the extracted DCN and the digested vector sequences. Bring up the Gibson Assembly options (Cloning → Gibson Assembly...). In the Backbone dropdown (Vector in Geneious R8 and earlier) select the digested vector if it is not already selected. Only the DCN should be shown in the Inserts box below.

Note that in Geneious 8.1 and above, the inserts and vector do not have to be pre-selected before opening the Gibson Assembly tool. Instead, they can be selected from within the setup options by clicking the Choose button.

Now check the other settings: The Min Overlap Length is set to a recommended value of 18bp. This is the length of the annealing bases, the complementary sequence part that two neighbouring sequences will have in common after the operation. When ligating two inserts, each will have half of this length as primer extension. In our case we're ligating one sequence into a vector and because primers are only created for insert sequences the insert primer will have the full 18bp as extension. Min Overlap Tm (48°C) is the melting temperature of the complementary sequences. Expand the Tm Calculation panel. These settings are used for the extension as well as the primer binding site (both get calculated independently). Most labs are using Phusion Polymerase, for which the recommended Tm gets calculated after a formula invented by Breslauer et al., so select this option under Formula. Confirm this dialog by clicking OK.

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A small dialog will indicate the progress, it should be finished within only a few seconds. A new Gibson Report document will appear in the Sequence Viewer. In the Hints section you will see a notice that the 5' overhanging bases that were created by the NcoI digestion have been removed from the sequence.

The most important part for now is the Generated Primer table. Primers are designed to bind to the Insert sequence, with the extension homologous to the flanking vector sequence. In the table you will see that the forward primer () contains the extension for the left flanking sequence, and the reverse primer () has an extension for the right flanking sequence. If you look on the reverse primer extension in green you will notice the 'cat' bases (highlighted in red in the screenshot) - these will be the template for the start codon we inserted before. As this primer is a reverse primer the start codon is shown as the reverse complement of ATG. The total length of the extension has been extended by the length of the modification, so that the complementary bases between the insert and the vector are still 18 bp long.



To check the results we can click on the link to the Product in the overview table above. The DCN CDS has been inserted before the GFP CDS and is flanked by both primers. When you hover over the primer annotation you will see the characteristics of the primer, the binding sequence and the extension sequence.



Scrolling in a bit further to the transition of the insert to the GFP we will notice the inserted ATG which is annotated as a manually inserted sequence. The translation shows this as a methionine and the GFP CDS is perfectly in frame.



You may want to check whether the melting temperatures for the primers are ok before you order them.

Congratulations, you successfully simulated a Gibson Assembly within Geneious.


Exercise 2: Advanced Batch-Cloning