Team:Rice University/CONSTRUCTS

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Revision as of 03:39, 30 October 2008

OUR TEAM:::SUMMARY ::: BACKGROUND ::: STRATEGY ::: CONSTRUCTS ::: RESULTS ::: ONGOING WORK

Constructs

Saccharomyces cerevisiae is widely used for baking and brewing, a versatile eukaryotic model system, and particularly useful for synthesizing metabolites under fermentation conditions. The microaerobic conditions of fermentation impede the oxidation of sensitive bioreactive compounds and are optimal for the de novo synthesis of resveratrol. To achieve our project goals and expand the yeast synthetic biology toolbox, we have constructed BioBricks encoding 3 yeast promoters, 3 yeast terminators, a two micron origin of replication, 2 selectable markers, 2 metabolic enzymes, and a yeast integration plasmid. We have also 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 Promoters

BBa_K122000	pPGK1	1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation.	1497
BBa_K122002	pADH1	700bp upstream of ADH1 promoter region. Constitutive promoter.	701
BBa_K122017	pGAL1 + tetO	Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR.	484

Yeast Terminators

BBa_K122003	tCYC1	300bp downstream the CYC1 coding region in a standard yeast strain.	300
BBa_K122004	tADH1	300bp downstream the ADH1 coding region in a standard yeast strain.	300
BBa_K122013	tPGK1	1000bp downstream the PGK1 coding region in an industrial yeast strain.	1000

Selectable Markers

BBa_K122018	ZeoR	Zeocin/Bleocin Resistance Gene	300
BBa_K122008	BleoR	Bleocin Resistance Gene under pTet promoter	800ish
BBa_K122014	ORI+HisTag	2 Micron ORI and auxotrophic histadine marker	<9000

Project Specific Constructs

BBa_K122001	[pGAL1][tetO][ZeoR]	874
BBa_K122005	Tyrosine Ammonia Lyase	1933
BBa_K122010	4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein	4000
BBa_K122012	[pPGK1][4CL:STS][tCYC1]	5497
BBa_K122015	[pGAL1][tetO][ZeoR][tADH1]	1175
BBa_K122021	[pADH1][TAL][tPGK1]	2651
BBa_K122019	[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]	1824

Additional Bacterial Parts

Novel Zero Leak Inverter (K122007 and K122006)

Amber Suppressor tRNA

K122007	The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine.	211
K122006	Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.	923

Through incorporation of amber stop codons or point mutations of tyrosine codons (TAC) to TAG within the coding region, a genetic circuit can be used to add an additional level of regulation and determine whether a full protein or partial peptide with be synthesized. This design has high signal-to-noise ratio, with virtually no leaky expression.

The Amber suppressed Red Florescent Protein (ARFP) has no virtually expression in SupF- (2) cells while visually apparent levels of red pigment is present in SupF+ cells (4). ARFP expression was compared against wtRFP expression in both cell types (1 and 3).

OUR TEAM ::: SUMMARY ::: INTRODUCTION ::: STRATEGY ::: RESULTS ::: ONGOING WORK ::: GALLERY

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        <tr>
          <td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td>
-         <td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons.</td>
+         <td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons.  Charges with tyrosine.</td>
          <td width="30">211</td>
        </tr>
        <tr>
          <td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td>
-         <td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the fluorophore.</td>
+         <td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.</td>
          <td>923</td>
        </tr>