From 2008.igem.org


Dear iGEM Judges,

I'm writing on behalf of the 2008 Cambridge iGEM team. Much of our work this summer involves investigating the use Bacillus subtilis as an iGEM chassis. In order to do so, we are creating a new set of Bacillus-compatible ectopic and integrational BioBrick plasmids. These plasmids combine ORFs from various sources, including homology regions to facilitate chromosomal insertion, a gram-positive replication origin, a BioBrick backbone, and standard PCR sites. They will contain appropriate Biobrick cut sites and scars, and the sequences are therefore indistinguishable from the existing standard.

However, to efficiently assemble these 'Frankenstein' vectors we utilized the In-Fusion method from ClonTech [1]. In-Fusion uses 15 base pair regions of homology between the ends of linear DNA fragments to join them together. This approach has many advantages over traditional digestion/ligation, including the ability to assemble as many as 4 or 5 pieces together in a single step. You can see a schematic of the approach on our wiki [2].

The In-Fusion method only complements the BioBrick standard, and the majority of our constructs this year were generated using traditional ligation. However, the ability to rapidly and easily assemble multiple fragments at once has been invaluable, and as the cost of oligos continues to fall, we believe this new assembly method will become increasingly attractive to Synthetic Biology.

While this method is currently proprietary and somewhat cost-prohibitive, one of our long-term focuses is to create an Open-Source version that can be widely adopted for future iGEM teams.

We look forward to sharing these vectors with the iGEM community and speaking about their assembly during our presentation at the Jamboree. We hope you will accept these new vectors for the competition this year.

Thank you,

Daniel Goodman and the University of Cambridge iGEM Team

[1] http://www.clontech.com/products/detail.asp?product_id=162275&tabno=2

[2] http://openwetware.org/wiki/IGEM:Cambridge/2008/Notebook/Bacillus



Thank you for your detailed email. Request approved. Please carefully document all differences from the BBa standard in your submissions and online materials, and how the two methods are related / compatible (and when they are not).

Also, we would be grateful if you could provide a brief written summary in some distributable format for how the team believes an open version of the method you described should be structured (i.e., standardized) so that we can begin to develop support for such an approach.

Best, Drew