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  1. Protein is Cash: Biomanufacturing in High School Classrooms Mary Jane Kurtz NBC2 Consultant Former Biotechnology Teacher at Minuteman Career and Technical HS Lexington, MA Presented at STEM Conference Indianapolis, IN Oct 2011

  2. Biomanufacturing/Biotechnology A plus for Teachers and Students • Integrated science education: Science Technology Engineering and Math (STEM) • Career pathways towards work/school focused on science with thousands of new jobs predicted in the next few years • Laboratory based activities = increased interest • More exciting ways of introducing concepts • Hands-on learning is more inclusive • State-of-the-art laboratories

  3. Biotechnology vs Biomanufacturing • Many high schools have adopted biotechnology as a part of the biology curriculum ie AP Biology • Biotechnology is DNA based with basic research as its focus • Career outcomes in biotechnology require four year BS degrees • Integrated with computer skills in areas such bioinformatics

  4. Biomanufacturing Curriculum: Standardized Concepts covered • Math,Biology,Chemistry present • Measurement • Solutions • Enzyme reactions • Transformation of cells with DNA • Forces used in centrifugation, electrophoresis etc. • National Academy of Sciences standards • Unit of Math & Science • Structure/properties of matter • Chemical reactions/conservation of matter • Cell structure and functions • Motions and forces

  5. Career Awareness • Begins with an interest in science at elementary levels • Shows the student how science is linked to future careers at the high school level • Directs students to pursue science in college and explore different area • Biomanufacturing should be one of these areas

  6. Protein is Cash/Workshop Activities • Introduction to Metrology and Instrumentation • Transformation of cells with DNA : Research • Upstream Processing • Select transformed cells from transformation • Grow transformed cells to optimal numbers • Downstream Processing • Purification of protein by cell centrifugation,lysis • Separation of desired protein by chromatography • Quantitative Analysis • PAGE analysis of proteins in column fractions

  7. Protein is Cash Workshop introduces the following career tracks: • Discovery Research • Validation of equipment • Transformation of cells with pGLO DNA plasmid • Upstream Processing • Production of pGLO into protein by transformed cell • Downstream Processing • Separation of cellular debris and cell supernatant • Purification of pGLO protein by Chromatography • Quality Control • Identification of protein product by electrophoresis

  8. Careers in Biomanufacturing

  9. Biomanufacturing • Based on production of needed substances by bioengineered organisms • Involves understanding of biology, chemistry, math, engineering and computer skills • Career outcomes can be found at several levels including degrees at high school, associate, bachelor in science, engineering, master and Ph.D.

  10. Day 1: Metrology/Instrumentation Activities • Calibration of top balance • Verification of pipette performance • Pipetman Challenge • Introduces good manufacturing practices (GMP) • SOP/Documentation • Confidence in measurements made by instrumentation Outcomes

  11. Day 2: Transformation of Bacteria Activities Outcomes Selection of cells by growing on ampicillin plates Only transformed with cells will survive due to amp-r gene Selected colonies will be grow in Luria broth mixture Aseptic technique and proper disposal • Addition of pglo plasmid to bacteria in Ca++ solution • Heat /shock the mixture • Plate cells onto selective Luria broth agar + ampicillin • Incubate overnight at 37oC • Note: arabinose acts to turn on production of pGLO protein

  12. Genetic Transformation in Ecolab Ampicillin resistance gene (Ampr) and target gene on bacterial plasmid Individual colony is selected and cultured to amplify recombinant DNA Plasmid enters some bacteria Only E. coli containing plasmid survive on Ampicillin plates Cell division Transformation mixture is plated on to agar plate containing Ampicillin Bacterial clones

  13. Results ofInserting Foreign DNA into an Organism • Cells will multiply and produce desired gene product • pGlO gene expression vector: Green Fluorescent Protein

  14. Upstream Processing Fermenter

  15. Day 2: Upstream Processing: Cell Collection and Lysis Activities Outcomes Multiplication of cells Initial separation of fluorescent protein • Transformed cells grown overnight in selected media are separated from media by centrifugation • Media is removed and packed cells are lysed • Homogenate is centrifuged • Supernatant with pGLO protein is retained for next step (glowing protein)

  16. Day 3: Downstream Processing: Purification of Green Fluorescent Protein by Chromatography Activities • Separation of product by Different types of Chromatography • Size Exclusion • Hydrophobic Interaction • Ionic Interaction Outcomes • Fractions with green fluorescent protein will glow and be selected for analysis • Understand concept of chromatography as selective interactions of compounds with matrix

  17. Sephadex Chromatography Red molecule =10^6 daltons - Blue molecule = 600 daltons

  18. Hydrophobic Chromatography H+ High salt 2 M (NH4)2SO4 H+ H+ H+ Wash buffer 1.3 M (NH4)2SO4 Low salt High Salt Elution Buffer 10 mM Tris Buffer

  19. Proteins bind to opposite charges on the matrix Addition of increasing Salt/pH should release proteins Ion Exchange Chromatography

  20. Day 4: Quality Control Activities Outcomes Analysis of protein samples by observation on light box determines purity Standard molecular weight markers indicate size of protein Verification of mol wt by comparison with standard proteins and number of proteins in a single sample • Chromatography fractions prepared for electrophoresis • Electrophoresis Box is assembled with PAGE gel • Samples of chromatography fractions are added to PAGE gel and ran for 30 minutes • Gels stained and viewed

  21. Quality Control Analysis of Column Fractions • Isolation and purification of GFP using Ion Exchange chromatography will be our focus • Electrophoresis by SDS PAGE of fractions collected.

  22. Isolation and Purification of Green Fluorescent Protein Chromatography Columns Transformed cells Test tubes #1 #2 #3 Fraction number

  23. Courtesy of ThermoScientific

  24. PAGE of pGLO samples after HIC chromatography A = molecular ladder B = no sample C = IEX #1 fraction column D = I EX # 2 fraction column E – H = wash fractions I = Supernatant J = molecular ladder K = standard GFP A B C D E F G H I J K

  25. Day 5. Clinical Trials and Food and Drug Administration Activities Outcomes Direct information about the industry Game for Clinical Trials provides hands-on activity for students that involves research and improved retention of concepts • Visit local biomanufacturing facility and or industry member presentation • History of FDA presented • Clinical Trials game for high school students

  26. Protein is Cash online

  27. Additional information about the biomanufacturing Field