Team:Rice University/STRATEGY

(Difference between revisions)

Revision as of 01:10, 30 October 2008

[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=169745 Tyrosine Ammonia-Lyase] (TAL, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]) - TAL catalyzes the conversion of L-tyrosine to p-coumaric acid and ammonia. TAL also exhibits Phenylalanine Ammonia-Lyase (PAL) activity, converting L-phenylalanine to trans-cinnamic acid and ammonia. Our work has focused on using [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=36575&Template=fungiYeast Rhodotorula glutinis] TAL because its ratio of TAL to PAL activity is high compared to other TAL homologs. In addition, previous studies have shown that this enzyme can be expressed as a functional protein in Saccharomyces cerevisiae and Escherichia coli. While the p-coumaric acid produced by TAL will serve as a substrate for resveratrol biosynthesis, the trans-cinnamic acid is expected to add a "floral" and "honey-like" bouquet to the beer.

[http://www.rcsb.org/pdb/explore.do?structureId=1Z1F Peanut STS] monomer bound to resveratrol.

[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_001084228.1 4-coumarate CoA ligase] :: [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=nuccore&term=&query_key=18&dopt=gb&dispmax=20&page=1&qty=1&WebEnv=1BAyWdNufvUWxpNM-oKD-9JoRgEPTXzU_kF02A2hfcePWB3nxyPvHO3gqlDJksRdZq9GmTDHNDmKEFuabzP4VKB%40263F5D1B8F754EA0_0062SID&WebEnvRq=1 Stilbene Synthase] Fusion Protein (4CL:STS, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]) - This enzyme fusion is comprised of Arabidopsis thaliana 4-coumarate-CoA ligase (4CL), which catalyzes the conversion of p-coumaric acid to 4-coumaroyl-CoA, and Vitis vinifera Stilbene Synthase, which catalyzes the condensation of resveratrol from 4-coumaroyl-CoA and three malonyl-CoA molecules. This 4CL:STS fusion protein was selected for our project because it has been shown to more efficiently produce resveratrol than coexpression of the proteins separately (possibly due to substrate channeling).

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122020 BBa_K122020].

Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and highly express the resveratrol pathway under anaerobic conditions.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 Cassette 1] - anaerobically induced expression of 4CL:STS
1. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 pPGK1] - serves the dual purpose of 5' homologous recombination region, and anaerobically induced promoter.
2. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 4CL:STS] - converts p-coumaric acid and 3 malonyl-CoA molecules to resveratrol.
3. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 tCYC1] - bi-directional transcriptional stop.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 Cassette 2] - inducible expression of Bleocin resistance.
1. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 pGAL1 TetO ZeoR] - provides galactose inducible, TetR repressible, Zeocin/Bleocin resistance. This part will allow us to select for recombinants.
2. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 tADH1] - bi-directional transcriptional stop. Strategically placed downstream of Zeor and upstream of pADH1 to prevent erroneous recombination at those regions.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 Cassette 3] - constitutive expression of TAL
1. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 pADH1] - constitutive promoter. placed strategically to prevent erroneous recombination with tADH1.
2. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 TAL] - converts L-tyrosine to p-coumaric acid and L-phenylalanine to trans-cinnamic acid.
3. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 tPGK1] - dual purpose of 3' homologous recombination region and bi-directional transcriptional terminator.

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 === Circuit Design for Recombination in Yeast===
 [[Image:circuit.png|center|700px|thumb|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122020 BBa_K122020].]]
-*Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and express the resveratrol pathway highly under anaerobic conditions.
+*Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and highly express the resveratrol pathway under anaerobic conditions.
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