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
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000] |
pPGK1 |
1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. |
1497 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002] |
pADH1 |
700bp upstream of ADH1 promoter region. Constitutive promoter. |
701 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 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
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 BBa_K122003] |
tCYC1 |
300bp downstream the CYC1 coding region in a standard yeast strain. |
300 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 BBa_K122004] |
tADH1 |
300bp downstream the ADH1 coding region in a standard yeast strain. |
300 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 BBa_K122013] |
tPGK1 |
1000bp downstream the PGK1 coding region in an industrial yeast strain. |
1000 |
Selectable Markers
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018] |
ZeoR |
Zeocin/Bleocin Resistance Gene |
300 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122008 BBa_K122008] |
BleoR |
Bleocin Resistance Gene under pTet promoter |
800ish |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014] |
ORI+HisTag |
2 Micron ORI and auxotrophic histadine marker |
<9000 |
Project Specific Constructs
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122001] |
[pGAL1][tetO][ZeoR] |
 |
874 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005] |
Tyrosine Ammonia Lyase |
 |
1933 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010] |
4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein |
 |
4000 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012] |
[pPGK1][4CL:STS][tCYC1] |
 |
5497 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015] |
[pGAL1][tetO][ZeoR][tADH1] |
 |
1175 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021] |
[pADH1][TAL][tPGK1] |
 |
2651 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019] |
[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1] |
 |
1824 |
Additional Bacterial Parts
Novel Zero Leak Inverter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006])
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] |
The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine. |
211 |
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 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).
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