Team:Rice University/RESULTS

A. Genetic Engineering and Part Construction

Saccharomyces cerevisiae are widely used for baking and brewing, and they are particular useful for synthesizing metabolites under fermentation conditions which prevent the air oxidation of many useful compounds. To achieve our project and expand the synthetic biology toolbox for programming yeast, we have introduced into the iGem registry BioBricks encoding 3 yeast promoters, 3 yeast terminators, a two micron origin of replication, 2 selectable markers, 2 enzymes, and a yeast integration plasmid. In addition, we have generated seven constructs using these parts. Furthermore, we have submitted two additional parts representing a foundational tool, including a gene encoding an amber suppressed RFP biobrick for screening of SupF+ (Amber suppressor) genotype and an amber suppressor tRNA biobrick.

Yeast Regulator Biobricks

Three unique constitutive and repressible yeast promoters were introduced into the registry.

Four additional yeast terminators were introduced into the registry.

Selectable Markers

Project Specific Constructs

This year's project involved the integration of 4-coumarate:coenzyme A ligase/ stilbene synthase fusion protein, a tyrosine ammonia lyase, and a zeocin resistance selectable marker. For more information, please visit Strategy.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122001]	[pGAL1][tetO][ZeoR]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]	Tyrosine Ammonia Lyase
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]	4CL:STS
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012]	[pPGK1][4CL:STS][tCYC1]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015]	[pGAL1][tetO][ZeoR][tADH1]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021]	[pADH1][TAL][tPGK1]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019]	[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]

Additional Bacterial Parts

Novel Zero Leak Inverter Concept

Please visit our Notebook for a summary of labwork, protocols, and overviews of progress.

B. Yeast Transformation

C. HPLC Data

To analyze our beer samples for resveratrol content, be will be using High Performance Liquid Chromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.

Figure C1. Example HPLC chromatogram of a resveratrol standard.

HPLC: Calibration

HPLC: Wine Extract Tests

HPLC: Fermenation batches

Fermentation

Coming Soon. For a sneak preview, check out the Gallery