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
RESEARCH &
DEVELOPMENT(pre-clinical):
Discovery Research
OPERATIONS:
Process development, Manufacturing
& Production
QUALITY:
Quality Control
& Assurance
CLINICAL RESEARCH:
Clinical Research:
Regulatory
Affairs
Senior Scientist
Scientist III,II,I
Research Associate
Process
Development
Director Supervisor &
Process Development
Technician
(QC)
Clinical Research
Clinical Research Manager
Bioinformatics
Scientist
Engineer
Analyst
Programmer
Manufacturing
Supervisor
Associate
Technician (Operator)
Instrumentation Tech
Calibration Technician
Facilities Management
Manager
Facilities Technician
Shipper/receiver
Chemistry
Chemistry QC
QC technician
Microbiology
QC analyst
QC Technician
Quality Assurance
(QA)
Documentation
Specialist
QA Documentation Coordinator
Regulatory Affairs
Data Manager
Business Development
Director of Business Development
Administration
Human resources
Safety Manager
Careers in red indicate
entry level positions
Entry level positions require an associates degree or certificate
Higher entry levels require a BS, MS, PhD or Engineering degrees

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
Outcomes
Introduces good manufacturing practices (GMP)
SOP/Documentation
Confidence in measurements made by instrumentation

11. Day 2: Transformation of Bacteria
Activities
Outcomes
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
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

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
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)
Multiplication of cells
Initial separation of
fluorescent 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
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. Ion Exchange Chromatography
Proteins bind to opposite charges on the matrix
Addition of increasing
Salt/pH should release proteins

20. Day 4: Quality Control Activities Outcomes
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
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

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
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
Visit local biomanufacturing
facility and or industry member presentation
History of FDA presented
Clinical Trials game for
high school students
Direct information about the industry
Game for Clinical Trials provides hands-on activity for students that involves research and improved retention of concepts

26. Protein is Cash online

27. Additional information about the biomanufacturing Field