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Introduction The Future
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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
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Introduction Common name Baker’s yeast and Brewer’s yeast (Campbell, 2002)
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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
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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
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Introduction
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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
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Introduction GRE3
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Project overview Step 1: Construction of plasmids
Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
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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 PGK1t URA3
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CONT…… Isolate xyn2 with PCR from previous study XYN2
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CONT…… Isolate xyn2 with PCR from previous study
PCR product digested with PacI and Ascl cloned in uPGK1 plasmid to make uPGK1X plasmid XYN2 PGK1t G418 URA3 PGK1 URA3
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Cont….. Plasmid used as a base for construction of the five expression vectors
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Cont….. 5.183kb 5000 3000 2000 6000 8000 1000 500 1500 3500 750 4000 10000 2500 4.1kb 0.51kb 1.8kb Fig 1b: Confirmation of the uENO1X plasmid by restriction enzymes Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes
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Cont….. 5.183kb 0.52kb 4.1kb Fig 1b: Confirmation of the uENO1X, uGAL10X plasmid by restriction enzymes 1.8kb Fig 1a: Confirmation of the uPFK2X plasmid by restriction enzymes
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Cont….. 5.2kb 0.57kb 4.1kb 1.8kb Fig 1b: Confirmation of the uENO1X, uGAL10X and uENO2pX plasmid by restriction enzymes Fig 1a: Confirmation of the uPFK2pX plasmid by restriction enzymes
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Cont…… 5.1kb 5.6kb 0.51kb 0.59kb Fig 2b: Confirmation of the uADH2X
Plasmid with REN 5.6kb 0.59kb 5.1kb 0.51kb Fig 2a: Confirmation of the uPGK1X plasmid with REN
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Project overview Step 1: Construction of plasmids
Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
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Project overview Step 1: Construction of plasmids
Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
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Method and Results Step 2:Transformation and Strain confirmation
Transformation in S. cerevisiae (Cho et al.,1999) Transformants selected on geneticin (G418)
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Cont….. NotI NotI
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Cont….. Strain confirmation - PCR confirmation - Using xyn2 primers
10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1000 610bp 750 500 250
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Cont….. RBB-xylan..xylose RBB-xylan..glucose RBB-xylan..galactose
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Project overview Step 1: Construction of plasmids
Step 2: Transformation and strain confirmation Step 3: xylanase activity assay Step 4: Growth curve, evaluation growth condition
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Many thanks Dr La grange Van Zyl lab (Stellenbosch university)
Acknowledgement Many thanks Dr La grange Van Zyl lab (Stellenbosch university) BMBT department (University of Limpopo) RSES for financial assistance
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Thank you ………………… OUR GOAL
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Introduction (Aristidou and Pentila,2000)
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