We’ve had a few MacVector users ask us recently if MacVector can be used to create constructs using the increasingly popular “Gibson Assembly” method. For those who are not familiar with this method, it was first described in 2009 by Daniel Gibson of the J. Craig Venter Institute. They showed that you can assemble multiple overlapping fragments in a single reaction to seamlessly join synthetic and natural genes to potentially create entire pathways or even genomes. Although they were originally using DNA fragments with relatively long overlaps (~450bp) to assemble large chunks of a genome, it has subsequently been shown that the reaction can be driven with overlaps as short as 20bp. This means that you can construct vectors from up to ten or more separate fragments (typically themselves generated by PCR with customized primers) in a single reaction, with no restriction enzymes required, as long as you design the fragments appropriately with unique 20+bp overlaps at each end. For more details on the technique there is a Wikipedia article and a University of Cambridge practical guide.
The key to emulating these constructs in MacVector is to create a “fake” restriction enzyme using the sequence of each overlap. You can then manipulate and join the overlapping ends using MacVector’s Cloning Clipboard functionality. Lets walk through a simple example so you can see how this works. Note that to get this to work as described, you MUST be using at least MacVector 12.7.4. You can download the update here.
For this example I’ll start with two cassettes – the first is for a tetracycline resistance gene;
and the second is for an ampicillin resistance gene;
You can see that the two cassettes share a 30bp overlap for which I’ve created a feature labelled as “overlapB” with a blue arrow head.
The first thing I’m going to do is create new fake restriction enzyme sites representing each of the three overlaps. While these could be created in a new “.renz” MacVector Restriction Enzyme file, I’m going to add them to the /Applications/MacVector 12.7/Restriction Enzymes/Common Enzymes.renz file. This is the default enzyme file that is used to display restriction enzyme cut locations in the Map tab of any opened DNA sequence. I can open the file in MacVector and then click on the Add button to drop down the enzyme editor sheet;
Next I switch back to my tet caassette sequence and select and copy the sequence corresponding to “overlapA”. Because I’d already created a feature all I have to do is click on the overlapA feature in the Map tab and then choose Edit | Copy. I can then switch back to the enzyme editor window and paste the sequence into the upper pane;
Here you can see that by default, the little arrows above and below the sequence are positioned at each end – this means that MacVector is treating the sequence as if it created a long 30 nucleotide 5′ overhang. While in reality this is a recombination sequence, not a restriction enzyme site, we will see later that this overhang is extremely useful as it only lets us join fragments that share the same recombination sequence. I can name the “site” and then repeat with overlapB and overlapC.
Finally I make sure that all three sites have their checkboxes selected in the list view;
The cassettes are automatically updated to show the new overlap “cut sites”;
I can now click on the overlapA site, hold down the shift key, select the overlap B site, click on the Digest button and the “cleaved” fragment appears on the Cloning Clipboard – here shown with the amp cassette digested in the same way;
I can then click on the right hand end of the tet cassette and drag a line – as I do that the compatible end of the amp cassette is shown with a black dot – incompatible ends are shown with a grey dot;
Finally, when I let go of the mouse over the target end, the two fragments are joined together;
Using this principle you can build up Gibson Assemblies from as many fragments as you like, then finally circularize the built-up vector when you have finished (assuming the last two ends are matching overlaps!). In addition, as you slowly build up a collection of overlap sequences in the restriction enzyme file, you can immediately identify those overlaps in any construct simply by opening the sequence and examining the Map view, or by running a full Analyze | Restriction Enzyme search using your edited .renz file.
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