From 2011.igem.org

As with the expansion of the iGEM competition, many iGEM teams have started to investigate the possibility of working with large-scale genomes. Large-scale gene manipulation often requires the use of tools which allow multiple gene inserts as to bring the cloning project from single gene level to a multiple gene level. However, the current BioBrick standard vectors available through iGEM are not designed for multiple-insert cloning. Therefore, the UW iGEM team decided to research methods to improve cloning efficiency and as a result, two "toolkits" were submitted to the registry.

Gibson Assembly Toolkit

To expand on work started by the 2010 UW IGEM team, this year we developed and submitted a set of plasmid backbones for BioBricks that are optimized for Gibson assembly. Based on the bglBrick standard [http://dspace.mit.edu/bitstream/handle/1721.1/46747/BBFRFC21.pdf?sequence=1 RFC 21], these pGA - plasmids for Gibson Assembly - vectors comprise the Gibson Assembly Toolkit. These vectors have much higher cloning efficiencies than the equivalent pSB vector and are fully compliant with BioBrick [http://www.synbio.org.uk/gibson/downloads/files/RFC57.pdf RFC 57] developed by the 2010 Cambridge iGEM team.

What's in the Gibson Assembly Toolkit?

• Five plasmid backbones
• 2 high copy vectors for gene extraction and cloning: pGA1A3, pGA1C3
• 1 medium copy expression vector: pGA3K3
• 2 low copy expression vectors: pGA4A5, pGA4C5

Magnetosome Toolkit

To demonstrate the utility of pGA vectorsIn addition, we were also ambitious about assembling a large gene-construct of over 16 kb. Therefore, utilizing the pGA vectors and Gibson cloning methods, we assembled the Magnetosome Toolkit with the goal to generate magnetic E. coli; a novel characteristic observed in magnetotactic bacteria such as Magnetospirillum magneticum.

What’s in the Magnetosome Toolkit?

• A set of the 18 essential genes for the various steps of magnetosome formation.
• Our favorite genes in pGA vectors
• A table compiling individual gene functions from our literature search