Laboratory in Biotechnology Process Engineering

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Laboratory in Biotechnology Process Engineering

• Susan C. Roberts
• Associate Professor, Chemical Engineering
• Director, Institute for Cellular Engineering, NSF
  IGERT Program

Preparing the STEM Workforce for the
Commonwealth Alternatives to Standard Laboratory
Courses
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Preparing 21st century STEM professionals

• MA industrial focus in biotechnology,
  pharmaceutical sciences and medical devices
• Workforce development and preparation
• Training on state-of-the-art robotics
  technologies
• Communication skills
• Interdisciplinary approaches life sciences and
  engineering/process sciences
• Recruitment retention
• Programs at the interface of engineering and the
  life sciences
• Innovative curricula student driven, problem
  focused, research based
• Excellent job placement statistics

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Overview

• Established with a Dreyfus Award in the Chemical
  Sciences
• Co-taught between Chemical Engineering and BMB
• 5050 representation of engineers and life
  scientists (senior level)
• Part of a two course sequence (lecture course in
  previous semester)

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Course design

• Groups of four students (two life scientists and
  two engineers) ? interdisciplinary mindset,
  teamwork, conflict resolution
• Oral and written presentations for each module ?
  communication skills
• Hypothesis driven with a focus on experimental
  design and data analysis ? problem solving and
  research approach
• Integration of high-throughput technologies ?
  training on state-of-the-art industrial equipment
  (first undergrad course in U.S. to use this
  technology)

Goal Production of a protein biologic following
a pharmaceutical industrial model
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Student driven and collaborative approach

• Student groups design experiments and choose
  conditions based on preliminary results and
  literature
• Pilot experiments inform large scale
  experiments
• Collect data and analyze as a class to determine
  optimal conditions in subsequent experiments at
  the chalk board
• Very interactive with a focus on interpretation
  of results and next directions
• Collaborative oral and written reports group
  leaders established and member contributions
  evaluated

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Integration of research

• Hardy Lab (Dept. of Chemistry) express human
  protein phosphatase 1 (hPP1) protein implicated
  in HIV and cancer
• Simple assay for activity exists
• Expressed in bacterial (E. coli) system
  (solubility is an issue)
• Purified using expensive/cumbersome
  equipment/methods
• Our goal - engineer an affinity tag onto hPP1 and
  inducibly express hPP1 in E. coli for
  large-scale production
• Can we
• Clone the gene into an appropriate E. coli
  expression vector?
• Express tagged hPP1 appropriately in E. coli?
• Increase the solubility of hPP1 to recover and
  retain activity?
• Better control the expression of hPP1 in E. coli?
• Improve the purity and yield of hPP1 using the
  affinity tag system?
• Project can be made fresh each semester using
  mutant forms of hPP1

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Industrial component

• Industrial facility tour (e.g., Vertex
  Pharmaceuticals, Cambridge, MA small molecule
  structure based drug design)
• Robotics equipment
• Product pipeline of drug discovery, synthesis and
  testing to identify lead compounds for clinical
  evaluation
• Interaction with lead scientists regarding
  science, economics and scale of operation
• Industrial advisory board of Chemical Engineering
  Dept. and Institute for Cellular Engineering
  (ICE) focus on relevant skills
• Alumni interaction with students
• Teach students skills relevant to industry (e.g.,
  lab notebooks, GMP, high-throughput technology,
  etc.)

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Student feedback (all positive)

• Decision making
• Collaborative work (e.g., discuss results as a
  class)
• Co-instructor model with high level of
  interaction amongst students and instructors
• Preparation for work in the pharmaceutical
  industry (several students said they would not
  have gotten the jobs they have without the
  experience in this course)
• Students enjoyed and learned from the
  troubleshooting that we did each day in lab
• Ability to work with students from other majors
• Experimental techniques learned are fundamental
  for any bioengineering project
• Understanding of the big picture how a
  process is designed and implemented
• Transition from small scale to large scale

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Summary

• Truly interdisciplinary laboratory course teach
  students to cooperate and communicate across life
  sciences and engineering disciplines
• Emphasis on experimental design and
  student-driven approaches
• Focus on communication (oral and written)
• Possibility for two concurrent sections with
  undergraduate and graduate students with new ISB
• Research based select a new protein each year
  so that course remains fresh
• Continue to increase industrial involvement and
  emphasis interaction with ICE
• Use as recruiting and retention tool

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Module 1Molecular Cloning of Target Gene

• Subclone a gene of interest into a suitable
  expression vector for large scale production
• Host expression system selection
• Expression plasmid considerations and design
• Directional cloning strategies
• Techniques PCR, DNA gel electrophoresis,
  restriction digestion ligation, bacterial
  transformation screening
• OUTPUT expression strain to be used in
  fermentation process

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Module 2Protein Expression and Fermentation

• Perform pilot experiments to determine best
  fermentation conditions at small scale
• Inducible vs. constitutive expression
• Enzyme activity assays
• Effect of environmental conditions
• Temperature, pH, cell density, inducer
  concentration, carbon source, etc.
• Design and conduct controlled fermentation
  experiment informed from pilot experiment results
• Operation of New Brunswick fermentor
• Growth kinetics
• OUTPUT fermentation broth for purification

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Module 3Protein Purification

• Cell disruption
• Each group evaluates potentially suitable methods
  in a small scale experiment and decides on
  conditions for extraction from fermentation broth
• Protein purification
• Ammonium sulfate precipitation
• Gel filtration
• Affinity chromatography
• Calculate yield and relative activity to assess
  purification success
• OUTPUT purified protein for analysis

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Module 4Protein Separation and Kinetic Analysis

• Protein separations using gel electrophoresis
• Western blotting to detect specific protein of
  interest (antibody-antigen binding)
• Enzyme kinetics calculation of Km and vm
• Determine time frame for experimental data
  collection
• Determine suitable enzyme concentration
• Conduct experiments over a range of substrate
  concentrations
• Compare activity /- HIS tag (added to protein to
  aid in purification)
• OUTPUT protein characterization and overall
  assessment of project success