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Explore the performance of six promoters for protein expression in S. cerevisiae during xylose growth. Construct plasmids and assess xylanase activity to understand promoter efficiency. The study involves transformation, growth evaluation, xylanase activity assay, and strain confirmation steps.
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Introduction The Future
Evaluation of S. cerevisiae promoters during growth on xylose Presenter: L. Mande Supervisor: Dr D.C. La Grange Co-Supervisor: Prof I. Ncube & Prof W.H. van Zyl
Introduction Common name Baker’s yeast and Brewer’s yeast (Campbell, 2002)
Introduction • S.cerevisiae has been used for years in the production of recombinant proteins • S.cerevisiae is a widely used organism • - fast cell growth • - tolerate high ethanol concentration • - tolerate wide spectrum of inhibitors • - GRAS status • - well-characterised physiology & genetics
Introduction • Recombinant protein production linked to biomass • S.cerevisiae is a Crabtree-positive yeast and produces less biomass when cultivated on glucose • Xylose second most abundant sugar and does not cause Crabtree-effect • Wild-type strains of S.cerevisiae cannot utilize xylose as carbon source
Aim& Objectives Aim: Evaluate six promoters commonly used for protein expression in S.cerevisiae, during growth on xylose Objectives: Construction of six plasmids with ENO1, PFK2, PGK1, ENO2 , ADH2, GAL10 with T. reesei xyn2 gene as a reporter gene Transformation of the plasmid in xylose utilizing strain S.cerevisiae (sXI, XYL3 , gre3::ZEO) - Xylanase activity assay using DNS assay - Growth curve and evaluation of growth in bioreactor
Introduction GRE3
Step 1: Construction of plasmids Project overview Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
Method & Results Step 1:Construction of six expression vectors - PGK1p and PGK1t isolated from yeast plasmid - G418 selection on geneticin - Target expression cassette to URA3 locus - Expression cassette cloned into pUC19 G418 PGK1 URA3 URA3 PGK1t
CONT…… • Isolate xyn2 with PCR from previous study XYN2
CONT…… • Isolate xyn2 with PCR from previous study • PCR product digested with PacI and Ascl cloned in uPGK1 plasmid to make uPGK1X plasmid XYN2 G418 URA3 PGK1t PGK1 URA3
Cont….. • Plasmid used as a base for construction of the five expression vectors
Cont….. Fig 1b: Confirmation of the uENO1X plasmid by restriction enzymes 5.183kb 10000 8000 6000 5000 0.51kb 4000 4.1kb 3500 3000 2500 2000 1.8kb 1500 1000 750 500 Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes
Cont….. 5.183kb Fig 1b: Confirmation of the uENO1X, uGAL10Xplasmid by restriction enzymes 0.52kb 4.1kb 1.8kb Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes
Cont….. 5.2kb Fig 1b: Confirmation of the uENO1X, uGAL10X and uENO2pXplasmid by restriction enzymes 0.57kb 4.1kb 1.8kb Fig 1a: Confirmation of the uPFK2pX plasmid by restriction enzymes
Cont…… 5.1kb 5.6kb 0.51kb 0.59kb Fig 2b: Confirmation of the uADH2X Plasmid with REN Fig 2a: Confirmation of the uPGK1X plasmid with REN
Step 1: Construction of plasmids Project overview Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
Step 1: Construction of plasmids Project overview Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
Method and Results Step 2:Transformation and Strain confirmation • Transformation • Transformation in S. cerevisiae (Cho et al.,1999) • Transformants selected on geneticin (G418)
Cont….. NotI NotI
Cont….. • Strain confirmation - PCR confirmation - Using xyn2 primers 10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1000 610bp 750 500 250
Cont….. RBB-xylan..xylose RBB-xylan..glucose RBB-xylan..galactose
Step 1: Construction of plasmids Project overview Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
Acknowledgement • Many thanks Dr La grange • Van Zyl lab (Stellenbosch university) • BMBT department (University of Limpopo) • RSES for financial assistance
Thank you ………………… OUR GOAL
Introduction (Aristidou and Pentila,2000)