Team:British Columbia/Data

From 2011.igem.org

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===Our System===
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==Our System==
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[[File:ubcigem2011system.jpg]]
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==Data for our Favourite New Parts==
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===Data for our Favourite New Parts===
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===GAL galactose-inducible yeast promoter===
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<html><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517002"><b>Main Page</a> - Alpha-Pinene Synthase Gene, BBa_K517002:</b> The (-)-a-pinene synthase (PsTPS-Pin) was isolated from Sitka spruce and converts geranyl pyrophosphate to 83.4%(-)-alpha-pinene, 12.6%(-)-beta-pinene, 2.1% Linalool, 1.0% (-)-beta-phellandrene, 0.4% Camphene, and 0.4% myrcene. We characterized it 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, which confirmed alpha- and beta-pinene as products. </br></br>
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Promoter can be induced to express a marker gene (GFP) when exposed to galactose in the absence of glucose.
 
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<html>This promoter was taken from the <a href="http://www.addgene.org/yeast-gateway/">S. cerevisiae Advanced Gateway Destination Vectors</a>.</html>
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517003"><b>Main Page</a> - Beta-Pinene Synthase Gene, BBa_K517003:</b> The (-)-b-pinene synthase (PsTPS-Pin2) synthase is a monoterpene synthase isolated from Sitka spruce converts geranyl pyrophosphate to 70.5%(-)-beta-pinene, 29.5%(-)-alpha-pinene. (-)-b-pinene 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, which confirmed alpha- and beta-pinene as products. </br></br>
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===GPD constitutive yeast promoter===
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517000"><b>Main Page</a> - GAL galactose-inducible yeast promoter, BBa_K517000:</b> Promoter can be induced when exposed to galactose in the absence of glucose. We characterized this part by placing a reporter GFP gene downstream of the promoter and determined that GFP expression was up-regulated when the yeast was shifted to galactose media.</br></br>
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Promoter for constitutively high expression.
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</html>
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<html>This promoter was taken from the <a href="http://www.addgene.org/yeast-gateway/">S. cerevisiae Advanced Gateway Destination Vectors</a>.</html>
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===Data for Pre-Existing Parts===
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===3-Carene Synthase===
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<b><a href="http://partsregistry.org/Part:BBa_K118025:Experience">Experience</a> - Limonene Synthase Composite + Lac promotor, BBa_K118025 (Edinburgh, iGEM 2008):</b> This part was previously uncharacterized. We expressed it in C41 <i>E. coli</i> 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 produced, demonstrating that this part works!</br>
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</br>
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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.  
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<b><a href="http://partsregistry.org/Part:BBa_J63006:Experience">Experience</a> - Yeast GAL1 Promoter, BBa_J63006:</b> We characterized this part by placing a reporter GFP gene downstream of the promoter. However, using the same protocol that successfully characterized our newly submitted BBa_K517000 GAL promoter and BBa_K517001 GPD promoter, we were not able to obtain clear evidence of BBa_J63006 promoter induction under exposure to galactose. Instead, for induction by galactose, we recommend using the BBa_K517000 GAL promoter created by our team until more evidence of BBa_J63006 functionality is obtained.</br></br>
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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.
 
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The iGEM team aims to characterize the 3-carene synthase in vivo in ''S. cerevisiae'' yeast. The products wil be sent for GC-MS.
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<b><a href="http://partsregistry.org/Part:BBa_K115056:Experience">Experience</a> - <i>E. coli</i> Isopentenyl Diphosphate Isomerase, BBa_K115056 (TU Delft 2008):</b> 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.
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</br></br></html>
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'''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.'''
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===We've also characterized the following parts===
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===Cineole Synthase===
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<html><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517001"><b>Main Page</a> - GPD constitutive yeast promoter, BBa_K517001:</b> Promoter for constitutively high expression. We characterized this part by placing a reporter GFP gene downstream of the promoter and determined that GFP was expressed regardless of whether the yeast was in glucose, raffinose or galactose media.</html>
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===Alpha-Pinene Synthase===
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===Beta-Pinene Synthase===
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===ERG20===
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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. 
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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.
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'''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'''
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'''Reference: Norihiko Misawa. Pathway engineering for functional isoprenoids. 2011.01.002 22:1-7'''
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===erg20-2===
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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.
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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.
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'''Reference: Oswald M., Fischer M., Dirninger N., Karst F. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. 2007 413:421'''
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===IDI1===
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===HMG2===
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==Data for Pre-Existing Parts==
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<a href="http://partsregistry.org/Part:BBa_K118025:Experience">Experience</a> - Limonene Synthase Composite + Lac promotor, BBa_K118025 (Edinburgh, iGEM 2008):</br>
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<a href="http://partsregistry.org/Part:BBa_K118024:Experience">Experience</a> - Limonene Synthase Composite, BBa_K118024 (Edinburgh, iGEM 2008):</br>
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<a href="http://partsregistry.org/Part:BBa_I742111:Experience">Experience</a> - Limonene synthase gene with ribosome binding site, BBa_I742111 (Edinburgh, iGEM 2007):</br>
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<a href="http://partsregistry.org/Part:BBa_I742110:Experience">Experience</a> - Limonene Synthase 1 from Citrus limon, BBa_I742110 (Edinburgh, iGEM 2007):</br>
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<a href="http://partsregistry.org/Part:BBa_J63006:Experience">BBa_J63006: Yeast GAL1 Promoter</a></br>
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<a href="http://partsregistry.org/Part:BBa_K115056:Experience">Experience</a> - E. coli Isopentenyl Diphosphate Isomerase, BBa_K115056 (TU Delft 2008):
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</br>
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</html>
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Latest revision as of 03:56, 18 October 2011

Team: British Columbia - 2011.igem.org

Contents

Our System

Ubcigem2011system.jpg


Data for our Favourite New Parts

Main Page - Alpha-Pinene Synthase Gene, BBa_K517002: The (-)-a-pinene synthase (PsTPS-Pin) was isolated from Sitka spruce and converts geranyl pyrophosphate to 83.4%(-)-alpha-pinene, 12.6%(-)-beta-pinene, 2.1% Linalool, 1.0% (-)-beta-phellandrene, 0.4% Camphene, and 0.4% myrcene. We characterized it 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, which confirmed alpha- and beta-pinene as products.

Main Page - Beta-Pinene Synthase Gene, BBa_K517003: The (-)-b-pinene synthase (PsTPS-Pin2) synthase is a monoterpene synthase isolated from Sitka spruce converts geranyl pyrophosphate to 70.5%(-)-beta-pinene, 29.5%(-)-alpha-pinene. (-)-b-pinene 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, which confirmed alpha- and beta-pinene as products.

Main Page - GAL galactose-inducible yeast promoter, BBa_K517000: Promoter can be induced when exposed to galactose in the absence of glucose. We characterized this part by placing a reporter GFP gene downstream of the promoter and determined that GFP expression was up-regulated when the yeast was shifted to galactose media.

Data for Pre-Existing Parts

Experience - Limonene Synthase Composite + Lac promotor, BBa_K118025 (Edinburgh, iGEM 2008): This part was previously uncharacterized. We expressed it 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 produced, demonstrating that this part works!

Experience - Yeast GAL1 Promoter, BBa_J63006: We characterized this part by placing a reporter GFP gene downstream of the promoter. However, using the same protocol that successfully characterized our newly submitted BBa_K517000 GAL promoter and BBa_K517001 GPD promoter, we were not able to obtain clear evidence of BBa_J63006 promoter induction under exposure to galactose. Instead, for induction by galactose, we recommend using the BBa_K517000 GAL promoter created by our team until more evidence of BBa_J63006 functionality is obtained.

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.

We've also characterized the following parts

Main Page - GPD constitutive yeast promoter, BBa_K517001: Promoter for constitutively high expression. We characterized this part by placing a reporter GFP gene downstream of the promoter and determined that GFP was expressed regardless of whether the yeast was in glucose, raffinose or galactose media.

Retrieved from "http://2011.igem.org/Team:British_Columbia/Data"