We explored hundreds of different limonene synthase genes that have been fully sequenced and documented. The search had to be refined down to only the S enantiomer of limonene, because the R enantiomer (which smells like turpentine), is not the chemical that had the properties needed for our project. We found three specific enzymes from lavender, Japanese catnip, and lemons that we elected to use for our project.
After locating specific limonene synthase genes that we were interested in using, we codon optimized the one from lemons. The codon optimization was critical because this gene normally exists in plant species, and we wanted to ensure we would have optimal expression using specific codons for Thermococcus kodakarensis. The next step was to create a promoter that would allow for replication in Escherichia coli and T. kodakarensis. This proved to be a challenge, considering our organism of choice does not have extensive research and has never been utilized for any other iGEM projects. After establishing a functioning promoter and Shine-Dalgarno sequence into the limonene synthase genes, we moved towards cloning into pLC71.
The pLC71 plasmid is the only plasmid in the world that will autonomously replicate in T. kodakarensis. We cloned the gene into the plasmid using restriction digestion technique at the NotI and SalI cut sites. After cloning, we confirmed that our sequence was correct and began to tackle the task of getting the plasmid into T. kodakarensis.
Using an anaerobic chamber to ensure our organism would not die, we used a heat transformation technique and plated our sample with applied selective pressure. The colonies that grew were then used to grow larger scale cultures that could be used for Gas Chromatography to detect the amount of limonene that was being produced. Different dilutions of limonene were used to determine the lower limit of detection before running our samples. After establishing the lower limit of detection, our samples were ran, but we did not see results conclusive enough to say limonene was present.
The team is currently working on troubleshooting the Gas Chromatography portion so we can detect the actual amounts of limonene being created. After returning from Boston, there will be continued work on the project by performing a western blot to confirm limonene synthase expression, protein purification of the limonene synthase gene for further analyzation, and determining how we can produce a higher yield of limonene using T. kodakarensis.