This year, we at the Cambridge team used Gibson Assembly exclusively for fabrication of our plasmids. Gibson assembly was pioneered in iGEM by the 2010 Cambridge team, who submitted an RFC (Request For Comments) to the BioBricks Foundation.
Teams at this year's iGEM competition have a very short time in which to complete their projects. We found that Gibson Assembly allowed us to construct our plasmids quickly and easily, saving us a lot of time.
See our protocol for Gibson Assembly here.
Gibthon, initiated by Bill Collins of the Cambridge 2010 iGEM team, is a collection of web based tools to facilitate the design of primers for Gibson Assembly. Currently, primer design is a bit of a 'dark art' – one must spend a large amount of time manually copying and pasting sequences into various other tools in order to check for annealing temperature, mispriming and secondary structure.
Gibthon's aim is to be an easy to use web based tool for primer design, reducing the chance of errors while designing primers. While some degree of common sense will always be required, Gibthon will reduce the chance of making mistakes while designing these primers.
Gibthon's source code is hosted on GitHub. You can see exactly what we have contributed this year by looking at my gibthon repository.
Our most important contributions are summarised below.
|Improvements to fragment display||Gibthon could only display fragments as raw text, making it difficult to decipher where features were on the strand.||Gibthon now displays fragments in a much more intuitive way, allowing the user to select parts of the sequence and easily copy from themm|
|Fragment import improvements||Gibthon had basic support for: partsregistry.org import (via an ugly hack), NCBI/Entrez import (by accession number only) and genbank file upload (much of the genbank metadata was lost)||Gibthon can now directly import from the partsregistry by part name (more below), seamlessly converting biobrick features and metadata into genbank format. It can also search a number of NCBI/Entrez databases, display search results, and import the results. Gibthon can now handle multiple file upload and supports both genbank and fasta format. You can now manually import raw sequences into Gibthon|
|Metadata Editing||Once a fragment was imported, it was impossible for the user to edit any of the metadata (annotations) associated with the fragment||Users can now easily update the metadata associated with a fragment – this is particularly useful, an example annotation might be “location: fridge C, shelf 2”|
|Fragment deletion||Gibthon did not allow you to delete a fragment from your fragment library||You can now|
Gibthon is an important tool not just for future iGEM teams, but for synthetic biology as a whole. Its open nature puts it in a great position to become the tool for construct assembly.
I am therefore intending to make further improvements after the wiki freeze, which will be:
- A few fixes for the fragment display (gibthon's UI is going through some improvements at the moment, so things may be a bit lopsided...)
- Great improvements to the Construct Designer.
- The construct designer (as is) works well, but it is not particularly intuitive for people new to the field (as many igemmers are) so I plan on making it far more visual – this will also make it harder for people to make mistakes!
- This will mean using the html5 canvas tag – People using IE6 will see a curt (but polite) explanation of why they should upgrade their browser!
We hope that gibthon will become a leading tool for construct design, both within iGEM and the wider synbio community. Gibthon will always be open source and free to use.
The Future of Gibson Assembly
We hope that more people in synbio will start using this revolutionary technique, it has certainly saved us a large amount of trouble and strife. While Gibson assembly does not require the prefix and suffix which are required for the BioBrick standard, it is perfectly straightforward to add and remove them using Gibson as required.
However, as the cost of synthesis comes ever lower, people will begin to move away from assembly – who wants to go to the hassle of building their construct if you can get the entire thing synthesised at little cost? When this begins to happen, physical DNA libraries will be less and less important, while tools to manipulate digital sequences of DNA will become vital.