Team:Rice University/CONSTRUCTS


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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 2-micron origin of replication, 2 selectable markers, 2 metabolic enzymes, and a yeast integration plasmid. We have also submitted two additional parts representing foundational tools, 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. 1497bp
BBa_K122002 pADH1 700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions. 701bp
BBa_K122017 pGAL1 + tetO Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR. 484bp

Yeast Terminators

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


Selectable Markers

BBa_K122018 ZeoR Zeocin/Bleocin Resistance Gene 300bp
BBa_K122008 BleoR Bleocin Resistance Gene under pTet promoter 80bp-ish
BBa_K122014 ORI+HisTag 2 Micron ORI and auxotrophic histidine marker <9000bp


Project Specific Constructs

BBa_K122001 [pGAL1][tetO][ZeoR] C2.jpg 874bp
BBa_K122005 Tyrosine Ammonia Lyase TAL.jpg 1933bp
BBa_K122010 4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein 4CL.jpg 4000bp
BBa_K122012 [pPGK1][4CL:STS][tCYC1] C1.jpg 5497bp
BBa_K122015 [pGAL1][tetO][ZeoR][tADH1] C2full.jpg 1175bp
BBa_K122021 [pADH1][TAL][tPGK1] C3.jpg 2651bp
BBa_K122019 [pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1] C1C2C3.jpg 1824bp


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. 211bp
K122006 Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation. 923bp

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).