Project Achievements
We searched through hundreds of different limonene synthase genes that have been fully sequenced and documented. The search had to be refined down to only looking for the S enantiomer of limonene, because the R enantiomer smells like turpentine. We were able to find three specific enzymes from lavender, catnip, and lemons that we chose 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 important because this gene normally exists in plant species, and we wanted to ensure that we would could get 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 little difficult, considering the fact that we are working with a very novel organism. After establishing a functioning promoter and a Shine-Dalgarno sequence into the limonene synthase genes, we moved towards cloning this into pLC71.
The pLC71 plasmid is the only plasmid in the world that will autonomously replicate in T. kodakarensis. We were able to clone 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 that our organism did not die, we used a heat transformation technique and then 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 created. Different dilutions of limonene were used to determine the lower limit of detection before running our samples. After establishing the lower limit of detection, we ran our samples and did not see conclusive enough results to say that limonene was being produced.
We are currently working on troubleshooting the Gas Chromatography portion so that we can detect the actual amounts of limonene being created. After returning from Boston, we will continue to work on our project by accomplishing a western blot to confirm limonene synthase expression, protein purification of the limonene synthase gene for further analyzation, and how we can produce a higher yield of limonene using T. kodakarensis..
