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Gibson Assembly Tutorial
Note: To complete the tutorial with the referenced data please download and install the tutorial above into Geneious Prime.
Gibson Assembly, also known as Gibson Cloning, is a method to assemble two or more linear fragments together without the use of restriction enzymes. Instead, the fragments have to be homologous at the sequence end (see image below, part (a)) so that they can ligate when a single strand is created. This principle is also found in various other related cloning methods like SLIC (sequence and ligase independent cloning (Li 2007)), CPEC (circular polymerase extension cloning (Quan 2009) & SLiCE (Seamless Ligation Cloning Extract (Zhang 2012)). The difference between each of the methods is how the single strand is created, either by melting the complete sequence (CPEC), excising with 3′ exonuclease activity (SLIC) or 5′ exonuclease activity (Gibson Assembly).
Gibson is an isothermal method where a T5 exonuclease is used to chew back the nucleotides from the 5′ end at 50°C (b). The T5 exonuclease is not heat stable, so it will get inactivated after a certain time (c). The overlapping homologous ends can then anneal, non-complementary parts get filled up with a polymerase (d) and the nick gets sealed with a ligase (e). The product is a scarless joint of the two initial sequences (f). Furthermore, this can be done with multiple sequences at once in one pot, allowing to create very large products with each cloning step.
This tutorial covers assembly of single and multiple fragments using Gibson cloning in Geneious. The Gibson cloning tool allows you to simulate your Gibson reaction and will produce a list of the PCR primers required to create the homologous ends.
Exercise 1: 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.
In order for this addition to be correctly incorporated into the primer extension, it needs to be annotated with a specific “Gibson Primer Extension” annotation. To add this, select the ATG bases and click the add primer icon.
Give the annotation a name, and add “Gibson Primer Extension” as the Type (you will need to type this in as it does not appear in the drop down list of types). Click OK and then Save the changes to the document (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 TAA (for example by clicking on the CDS annotation, then holding Shift+click just before the ‘TAA’). We’re going to extract this without the use of primers, as they will be generated later by Geneious during the Gibson operation. Click on Extract in the Sequence Viewer and chose an appropriate name, such as “DCN CDS”.
Now select both the extracted DCN CDS and the digested vector sequences. Bring up the Gibson Assembly options (Cloning → Gibson Assembly…). In the Backbone dropdown select the digested vector if it is not already selected. After the vector is selected as the backbone, the tag representing this sequence in the Construct Layout panel should turn green, and the insert tag should be grey, as in the screenshot below. Click the tags to see a preview of the sequences that will be used in the assembly.
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.
Check that the Exonuclease is set on 5′ Exonuclease. If you are using a related method that uses a different exonuclease (such as SLIC or In-Fusion cloning), you can choose this here.
Now click Primer Options to check the primer settings. The Min Overlap Length is set to a recommended value of 18 bp. This is the length of the annealing bases, the complementary sequence part that two neighboring 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 18 bp as extension. Min Overlap Tm (48°C) is the melting temperature of the complementary sequences. The Tm 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, then click OK in the main Gibson Assembly options to run the analysis.
Once the assembly has run, a new folder containing the Gibson Report, ligated sequence product, and generated primers will appear in the Document Table. Open the Gibson Report. In the Hints section you will see a note that the 5′ overhanging bases that were created by the NcoI digestion have been removed from the sequence, and the bases we annotated as “Gibson Primer Extension” have added to the primer.
The Generated Primer table lists the primers required for this reaction, and the primers listed here also appear as separate files in the Document 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 are the reverse complement of the bases for the start codon that we added as a Gibson Primer Extension. The total length of the extension has been extended by the length of the added start codon, so that the extension is 21 bp long in total, but 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 Gibson Report. 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 okay before you order them.
Exercise 2: Advanced Batch-Cloning
Many experiments might require a more complex setup than only assembling two fragments to each other or inserting one sequence into a vector. Gibson Assembly in Geneious Prime handles batch operations by using sequence list documents. Each sequence in the list will be inserted into the same position, and a new product & corresponding primers will be generated for each sequence.
In this example we want to test the expression of the DCN fusion gene with a variety of different promoters.
Select the DCN CDS and digested vector from the first exercise, plus the sequence list ‘Promoter’. This is a list of 5 promoters downloaded from NCBI. Start the Gibson Assembly operation (Cloning → Gibson Assembly…)
This time you should see an additional tag for the Promoters in between the vector and insert sequences. If it does not appear in between the vector and insert, drag it to this position to ensure that the promoters are inserted 5′ of the DCN gene. The tag is a brown color, indicating that it is a sequence list instead of a single sequence. Because we’re expecting 5 products make sure you save them in a subfolder. The other settings should still be remembered from the previous run.
Click OK and open the Report Document in the subfolder after the operation has finished. This time we see all the five products listed after the ‘Hints’ section, with only the promoter (Insert 1) changing. We have more primers, and when scrolling down we find 4 primers that are greyed out. This is because these are the primers between the DCN CDS and the vector, and they are the same for all 5 products, so only one of them has to be ordered. For each product, a sequence list containing the primers required to generate that product should be present in the Document Table. When scrolling through the primers you might notice that some of them are pretty short – the shortest primer binding site is only 11 bases long. So pay attention to this as you may have to manually adjust these primers or consider using different promoter sequences.