Problem-Based Learning
Laboratories on
Chemicals from Biorenewables
Bioseparations
C. Glatz, S. Mallapragada, B.
Narasimhan, P. Reilly and J. Shanks
Department of Chemical Engineering
M. Huba
Educational Leadership and Policy Studies
Iowa State University, Ames, IA 50011-2230
Z. Nikolov
ProdiGene, Inc. and TAMU, College Station TX
Vision

We have developed four 1-credit open-ended,
multidisciplinary laboratory courses involving
“Chemicals from Biorenewables”. These problem-based
learning laboratories have been integrated with existing
and new bioengineering-related ChE classes

Target audience:
– undergraduate (seniors) and graduate students in
Chemical Engineering
– undergraduate and graduate students in
Biochemistry and Biophysics, Biology and Food
Science.
Motivation: Topic

ChE evolving from a petrochemical-based to a
biorenewables-based discipline. Examples:
Product
Indigo
poly(lactic acid)
Biopol
1,3 propanediol


Species used
Microbial
Microbial
Microbial/plants
Microbial
Company
Genencor
Cargill/Dow
Monsanto
DuPont
Current ChE curriculum does not reflect this trend
Introduce new courses to cover this new technology
Motivation: Educational

Problem-based learning
–
–
–
–

Open-ended problems
Learning-based approach
Students direct learning of the topic
Problems provide motivation for learning
Multidisciplinary
– Team-based approach

ABET criteria
– Life-long learning
Curriculum Structure





Four new 1-credit laboratories - each associated
with an existing or new ChE undergraduate/
graduate level biotechnology related theory
course
Each laboratory course has one open-ended
design project topic and list of desired outcomes
Students work in teams of three - each team has
a student with a biology/biochemistry
background
Opportunity for problem-based, studentdirected, multidisciplinary team-based learning
Bioethics component
General Lab Course Outline

First two weeks: Common component for all the lab
classes - Teach students statistics, bioethics, how to work
in teams, literature searches, laboratory notebooks.
Faculty member plays role of instructor with learning
exercises in context of technical content of the course.

Next three weeks: Literature review, coming up with
plan for solving the problem, team roles, some laboratory
training. Faculty member plays role of coach.

Next nine weeks: Implementation of plan,
experimental design. Faculty member plays role of coach

Last two weeks: Wrapping up, written and oral
presentations
Description of Laboratory Courses

Bioinformatics - (Spring 03: Reilly) - Development of
bioinformatic and virtual reality techniques for
investigating and predicting enzyme structure and
function.
 Metabolic Engineering - (Spring 02: Shanks) Combination of experimental methods with mathematical
analysis of the metabolism of ethanol fermentation from
yeast.
 Bioseparations - (Fall 02: Glatz) - Development of a
process for recovering a recombinant protein expressed
in corn germ.

Tissue Engineering - (Fall 02: Mallapragada,
Narasimhan) - Development of a bioreactor to cultivate
bioartificial skin in vitro on suitable biodegradable
polymer scaffolds
Acknowledgments





NSF Combined Research and Curriculum
Development Grant EEC 0087696
Barry Lamphear and Susan??, Prodigene,
Inc. for assistance with ELISA.
Nicolas Deak, Erin Denefe and Tom Mathews
for their presentation.
Summer research crew of Danielle
McConnell, Jim Kupferschmidt, Yandi
Dharmadi, Zhengrong Gu, Maureen Griffin
Tutors Todd Menkhaus and Kevin Saunders
Brazzein Purification
ChE 562
12/06/02
Goal Objectives:
• Develop a separation process to recover Brazzein
from transgenic corn
• Purity must be > 80% of total protein content
• Salt content in final product must be less than
0.01M
Starting material:
• Defatted transgenic corn germ meal with some
endosperm contamination. Initial brazzein
concentration 250 g per gram of meal
Brazzein molecular structure:
Brazzein Information
•
•
•
•
•
•
Small molecule 6500 Da
Thermo-stable 32-82 C
Water Soluble pH= 3.6, 4, and 7
pI=5.4
Water solubility will be at least 50 mg/ml.
Two different types of brazzein. Type II twice
as sweet as Type I.
Experimental Procedures
Transgenic
Corn Germ
Extraction
Cation Exchange
Chromatography
Size Exclusion
Purified Brazzein
Size Exclusion
Extraction Variables
Protein/Water Ratio = 1g/6 mL, temperature = 23
pH= 4.0-5.5
Salt concentration =50 NaOAc: 100 mM NaCl
Mixing time of 45 minutes
Protein extraction vs pH
Protein concentration (ug/mL)
•
•
•
•
1800
1559.5
1600
1357.9
1400
1200
1000
800
594.4
690.7
600
400
200
0
pH 4
pH 4.5
pH 5
pH 5.5
Size Exclusion HPLC
• The objective for this test was to asses whether
simple membrane filtration was applicable in this
case.
• The Brazzein rich extract (pH 4) is eluted in a Size
Exclusion HPLC
• Elution conditions:
– pH 4.0
– NaAc Buffer ( NaAc 20mM, NaCl 30 mM)
• A standard elution curve was run in parallel, using
known MW markers.
HPLC Standard Curve
Molecular Weight (kD)
750
500
250
0
10
12
14
16
18
20
Time (min)
Molecular Weight = 147416 exp(-0.4751* Time)
R2 = 0.9404
22
24
HPLC Results
Contaminant
Brazzein
2.91 kDa
6.5 kDa
22.8 min.
21.1 min.
HPLC Conclusions
• A direct membrane filtration of our
Brazzein rich extract is not applicable in
this case
• The overlapping contaminant protein peak
is important enough to try some other
means to purify our product
Cation Exchange
Chromatography
• Goal: Separate brazzein from the 2.91 kDa
contaminant
• Cation exchange successful removing brazzein
from yeast cells (Irwin)
• Resin type: SP (sulphopropyl) Sepharose Fast
Flow
• Elute with linear salt gradient 0-1 M NaCl
Cation Exchange Setup
• Column: SP Sepharose fast flow resin
• Length = 8 cm
• Diameter = 1 cm
• Volume = 6.28 mL
• Buffers:
• A = 20 mM NaOAc, pH 4.0
• B = Buffer A + 1.0 M NaCl
• Operation:
• Flowrate = 1.0 mL/min
Cation Exchange for Pure
Brazzein
Sample 40 g/mL pure brazzein in DI water
Equilibration of column with Buffer A
Load of 10 column volumes
Gradient elution from 0%-100% Buffer B in
15 column volumes
• Isocratic flow of 100% buffer in 5 column
volumes
•
•
•
•
Cation Exchange for Corn
Germ
• Sample 20 g + 120 mL Buffer A
• Mixed 40 min, centrifuged 30 min. (15000
rpm), and filtered (0.22 m CA)
• Equilibration of column with Buffer A
• Load of 5 column volumes
• Gradient elution from 0%-100% Buffer B in
15 column volumes
• Isocratic flow of 100% buffer in 5 column
volumes
Cation Exchange Results
• Section of concern 27-43 minutes after elution
begins (30-45% Salt gradient)
• Estimate fold of purification
• assuming no nonprotein UV280 adsorbing
materials
• Perform experiment to determine molecular
weight of material in this section
1.8
100
1.6
Light Absorbance (UV)
1.2
Pure Brazzein
60
Nontransgenic Corn
1.0
Salt Gradient
0.8
40
0.6
0.4
20
0.2
0.0
0
-3
-2
-1
0
Time (hr)
1
2
Salt Gradient (% 20 mM NaOAc)
80
1.4
1.8
100
1.6
80
1.2
Absorbance Fraction
Pure Brazzein
60
Transgenic Corn
1.0
Salt Gradient
0.8
40
0.6
0.4
20
0.2
0.0
-3.0
0
-2.5
-2.0
-1.5
-1.0
-0.5
Time (hr)
0.0
0.5
1.0
1.5
Salt Gradient (% 20 mM NaOAc)
1.4
Conclusions:
• Cationic exchange chromatography is more
effective (72 %)
• This is not enough to comply with our
specifications
• After analyzing SDS-PAGE electrophoresis
results we decided to do a size exclusion
• Expected results will be within
specifications
Questions?
Do you have any suitable problem?
Impact


Make ChE education more relevant for our
undergraduate students
Teach students
– problem-based learning techniques
– develop their metacognitive abilities
– life-long learning


Coupling these educational techniques with
valued new technologies
Integrate some of these new experiments in a
non open-ended manner into the required ChE
undergraduate laboratories
Assessment

Self- and instructor-assessment using
– Teamwork rubric
– Design rubric
– Written report rubric
– Oral presentation rubric
Curriculum Structure
Biochemical
Engineering
Metabolic
Engineering
Upstream
Processing
Microbial
Engineering
Lab
Bioinformatics
Lab
Bioseparations
Polymeric
Biomaterials
Downstream
Processing
BioSeparations
Lab
Product
Development
Tissue
Engineering
Lab