Team:British Columbia/Data

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Contents 1 Our System 2 Data for our Favourite New Parts 2.1 GAL galactose-inducible yeast promoter 2.2 GPD constitutive yeast promoter 2.3 3-Carene Synthase 2.4 Cineole Synthase 2.5 Alpha-Pinene Synthase 2.6 Beta-Pinene Synthase 2.7 ERG20 2.8 erg20-2 2.9 IDI1 2.10 HMG2 3 Data for Pre-Existing Parts

Our System

Data for our Favourite New Parts

GAL galactose-inducible yeast promoter

Promoter can be induced to express a marker gene (GFP) when exposed to galactose in the absence of glucose.

This promoter was taken from the S. cerevisiae Advanced Gateway Destination Vectors.

GPD constitutive yeast promoter

Promoter for constitutively high expression.

This promoter was taken from the S. cerevisiae Advanced Gateway Destination Vectors.

3-Carene Synthase

The 3-carene synthase is a monoterpene synthase isolated from Picea sitchensis by Chris Keeling and Dr. Joerg Bohlmann. This 3-carene synthase was found to confer resistance to the white pine weevil, part of the reason why the iGEM team decided to characterize this part in yeast. This synthase converts farnesyl pyrophosphate to 66.4% (+)-3-Carene, 16.3% Terpinolene, 2.7% (-)-alpha-pinene, 2.5% Terpinen-4-ol, 2.1% (-)-beta-phellandrene, 2.1% myrcene, 1.4% alpha-Terpinol, 0.8% gamma-terpinene, 1% others.

The 3-carene synthase has been previously characterized by expression in C41 DE3 E. coli followed by a His-tag purification. The purified enzymes were assayed in vitro with GPP and sent for GC-MS.

The iGEM team aims to characterize the 3-carene synthase in vivo in S. cerevisiae yeast. The products wil be sent for GC-MS.

Reference: Keeling, CI, Weisshar S, Ralph SG, Jancsik S, Hamberger B, Dullat HK, and Joerg Bohlmann. Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.) BMC. 2011 March 7 11:43.

Cineole Synthase

Alpha-Pinene Synthase

Beta-Pinene Synthase

ERG20

The ERG20 gene is an essential gene naturally found in yeast. It encodes for an enzyme that assists the mevalonate pathway to produce FPP and GPP from I-PP and DMA-PP. Our iGEM team would like to produce more GPP than FPP because GPP is a precursor in producing monoterpenes where as FPP produces diterpenoids instead.

The iGEM team aims to characterize this gene in vivo in S. cerevisiae yeast. The products wil be sent for GC-MS. Furthermore, this will be made into a biobrick and become an addition to the iGEM parts registry.

Reference: Ignea C., Cvetkovic I., Loupassaki S., Kefalas P., Johnson C. B, Kampranis S. C, Makris A. M. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. 2011 10:4

Reference: Norihiko Misawa. Pathway engineering for functional isoprenoids. 2011.01.002 22:1-7

erg20-2

The erg20-2 gene is a mutant of the ERG20 gene. It is a mutant because of the K197 to E substitution. It also for an enzyme that assists the mevalonate pathway to produce FPP and GPP from I-PP and DMA-PP. It was observed that this gene produces "25% FPP and 75% GPP instead of 75% FPP and 25% GPP" when compared with the wild type ERG20 gene.

The iGEM team aims to characterize this gene in vivo in S. cerevisiae yeast. The products wil be sent for GC-MS. Furthermore, this will be made into a biobrick and become an addition to the iGEM parts registry. In particular, we would like to determine whether more monoterpenes are produced with this mutant gene than the wild type ERG20 gene.

Reference: Oswald M., Fischer M., Dirninger N., Karst F. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. 2007 413:421

IDI1

HMG2

Data for Pre-Existing Parts

Experience - Limonene Synthase Composite + Lac promotor, BBa_K118025 (Edinburgh, iGEM 2008): We expressed this part in C41 E. coli that possesses a pRARE plasmid that enables it to translate proteins with rare codons. The limonene synthase was purified from a culture and subjected to an enzymatic assay for its ability to synthesize limonene monoterpene from GPP substrate. GC-MS analysis showed that limonene was present in the sample where GPP was added. In contrast, the control without GPP did not contain limonene. This experiment was performed a second time and similar results were obtained. Other registry parts that are relevant to this part: Experience - Limonene Synthase Composite, BBa_K118024 (Edinburgh, iGEM 2008), Experience - Limonene synthase gene with ribosome binding site, BBa_I742111 (Edinburgh, iGEM 2007), Experience - Limonene Synthase 1 from Citrus limon, BBa_I742110 (Edinburgh, iGEM 2007).

BBa_J63006: Yeast GAL1 Promoter

Experience - E. coli Isopentenyl Diphosphate Isomerase, BBa_K115056 (TU Delft 2008): We sequenced this part using the VF2 and VR primers and found that the sequence between the original EcoRI and PstI restriction enzyme sites (1) did not contain the XbaI or SpeI sites and (2) was not homologous to any part of the IDI gene. We recommend that this part is removed from the registry and taken off distribution.