Teaching Biomanufacturing in High School Classrooms

Report
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
Biomanufacturing/Biotechnology
A plus for Teachers and Students
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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
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More exciting ways of introducing concepts
Hands-on learning is more inclusive
State-of-the-art laboratories
Biotechnology vs Biomanufacturing
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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
Biomanufacturing Curriculum:
Standardized Concepts covered

Math,Biology,Chemistry
present
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Measurement
Solutions
Enzyme reactions
Transformation of cells
with DNA
Forces used in
centrifugation,
electrophoresis etc.

National Academy of
Sciences standards
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Unit of Math & Science
Structure/properties of
matter
Chemical
reactions/conservation
of matter
Cell structure and
functions
Motions and forces
Career Awareness
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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
Protein is Cash/Workshop Activities
Introduction to Metrology and Instrumentation
Transformation of cells with DNA : Research
Upstream Processing
1.
2.
3.
1.
2.
Select transformed cells from transformation
Grow transformed cells to optimal numbers
Downstream Processing
4.
1.
2.
Purification of protein by cell centrifugation,lysis
Separation of desired protein by chromatography
Quantitative Analysis
5.
1.
PAGE analysis of proteins in column fractions
Protein is Cash Workshop introduces
the following career tracks:
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Discovery Research
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Upstream Processing

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Production of pGLO into protein by transformed cell
Downstream Processing
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Validation of equipment
Transformation of cells with pGLO DNA plasmid
Separation of cellular debris and cell supernatant
Purification of pGLO protein by Chromatography
Quality Control

Identification of protein product by electrophoresis
Careers in Biomanufacturing
RESEARCH &
DEVELOPMENT(preclinical):
Discovery Research
OPERATIONS:
QUALITY:
Process development,
Manufacturing
& Production
Quality Control
& Assurance
Discovery Research
Process
Development
Director Supervisor &
Process Development
Technician
Quality Control
(QC)
Bioinformatics
Scientist
Engineer
Analyst
Programmer
Manufacturing
& Production
Supervisor
Associate
Technician (Operator)
Instrumentation Tech
Calibration Technician
Facilities Management
Manager
Facilities Technician
Shipper/receiver
Chemistry
Chemistry QC
Analyst
QC technician
Microbiology
Microbiology
QC analyst
QC Technician
Quality Assurance
(QA)
Manager
Documentation
Specialist
QA Documentation
Coordinator
•Careers in red indicate
entry level positions
Entry level positions
require an associates
Higher entry levels
require a BS, MS, PhD or
Senior Scientist
Scientist III,II,I
Research Associate
CLINICAL RESEARCH:
Clinical Research:
Regulatory
Affairs
Clinical Research
Clinical Research
Manager
Clinical Research
Clinical Research
Manager
Regulatory Affairs
Manager
Associate
Data Manager
Business Development
Director of Business
Development
Administration
Human resources
Safety Manager
Biomanufacturing
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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.
Day 1: Metrology/Instrumentation
Activities
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Calibration of top
balance
Verification of
pipette performance
Pipetman Challenge
Outcomes
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Introduces good
manufacturing
practices (GMP)
SOP/Documentation
Confidence in
measurements made
by instrumentation
Day 2: Transformation of Bacteria
Activities
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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
Outcomes
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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
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
Cell division
Bacterial clones
Only E. coli containing plasmid
survive on Ampicillin plates
Transformation mixture is plated
on to agar plate containing
Ampicillin
Results ofInserting Foreign DNA into
an Organism
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Cells will multiply and
produce desired gene
product
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pGlO gene expression
vector: Green Fluorescent
Protein
Upstream Processing Fermenter
Day 2: Upstream Processing: Cell
Collection and Lysis
Activities
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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)
Outcomes
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Multiplication of cells
Initial separation of
fluorescent protein
Day 3: Downstream Processing:
Purification of Green Fluorescent Protein
by Chromatography
Activities
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Separation of product
by Different types of
Chromatography
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Size Exclusion
Hydrophobic Interaction
Ionic Interaction
Outcomes
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Fractions with green
fluorescent protein will
glow and be selected
for analysis
Understand concept of
chromatography as
selective interactions of
compounds with matrix
Sephadex Chromatography
Red molecule =10^6 daltons - Blue molecule = 600 daltons
Hydrophobic Chromatography
H+
H+
H+
H+
Low salt
High salt
High Salt
2M
(NH4)2SO4
Wash buffer
1.3 M
(NH4)2SO4
Elution Buffer
10 mM Tris
Buffer
Ion Exchange Chromatography
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Proteins bind to
opposite charges on
the matrix
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Addition of increasing
Salt/pH should release
proteins
Day 4: Quality Control
Activities
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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
Outcomes
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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
Quality Control Analysis of Column
Fractions
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Isolation and purification of GFP using Ion
Exchange chromatography will be our focus
Electrophoresis by SDS PAGE of fractions
collected.
Isolation and Purification of Green
Fluorescent Protein
Chromatography
Columns
Transformed
cells
Test
tubes
Fraction
number
#1
#2
#3
Courtesy of
ThermoScientific
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
Day 5. Clinical Trials and Food and
Drug Administration
Activities
 Visit local biomanufacturing
facility and or industry
member presentation
 History of FDA presented
 Clinical Trials game for
high school students
Outcomes
 Direct information about
the industry
 Game for Clinical Trials
provides hands-on activity
for students that involves
research and improved
retention of concepts
Protein is Cash online
http://biomanonline.com
Additional information about the
biomanufacturing Field
www.biomanufacturing.org
www.bio-link.org
www.biomanonline.com
www.fda.gov/cder/about/history/default.htm
www.drugbank.ca
[email protected]

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