We developed pHF10, a derivative of pLC71, the only known vector plasmid for Thermococcus kodakarensis, as a basis for our experimentation. PLC71 has several important features, including a ribosomal binding site and promoter for E-coli, allowing it to be purified via ordinary miniprep methods. The additions to our vector plasmid were a limonene synthase gene, a promoter optimized for use in kodakarensis, and selective markers for lovastatin resistance and tryptophan synthesis. The introduction of the gene block to the pLC71 plasmid required use of restriction enzymes and ligation techniques to insert the limonene synthase gene into the backbone.
To our knowledge, this is the first iGEM project to use the archaea Thermococcus kodakarensis. The iGEM standard vectors will not replicate within Thermococcus kodakarensis, so we chose to use a plasmid (pLC71) that would replicate in our organism to further the project.
No iGEM compatible plasmid backbones currently exist for kodakarensis, and though there are some parts designed for other archaea, they may or may not function correctly in this organism.
The usefulness of novel chassis is clear, and shows the need for new organisms and new backbone structures to be added to the iGEM database. Thermococcus kodakarensis and the pLC71 plasmid vector could be two such additions.