Team:Vanderbilt/Parts

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).

Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in S. cerevisiae. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.

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 (Tirabassi 2014).

pVU14006

As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks.

A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription.

BBa_K1322001- All-RFC Compatible Fluorescent Oscillator

As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th.

Sabinene Synthase

Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.

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.

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