Team:Vanderbilt/Parts

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BBa_K1322231- Optimized Santalene Synthase

BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.

The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,

In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).

We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Rebecca Tirabassi).

pVU14004

As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has .... pVU14006

BBa_K1322001- All-RFC Compatible Fluorescent Oscillator

Sabinene Synthase



References:
Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8.
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014. BBa_K1322231 BBa_K1322001