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Edvo-Kit #305
305
Fermentation and
Bioprocessing of GFP
Experiment Objective:
This experiment is designed to introduce the basic concepts of fermentation and bioprocessing
through the production of GFP protein in a small-scale fermentor. At the end of the activity,
students will observe and analyze results and will be able transform the abstract concepts of
fermentation and bioprocessing into an enhanced scientic understanding.
305.140822
Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
Table of Contents
Page
Experiment Components 3
Experiment Requirements 3
Background Information 4
Experiment Procedures
Experiment Overview and General Instructions 9
Laboratory Safety 10
Module I: Preparation of Seed Culture 11
Module II: Growth of GFP in the Fermentor 12
Module III: Purication of GFP Protein (Bioprocessing) 13
Study Questions 14
Instructors Guidelines 15
Overview 15
Pre-Lab Preparations 16
Study Questions and Answers 17
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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP
Experiment Components
Component Check ()
Microcentrifuge tubes
Syringe
15 ml Centrifuge tubes
Transfer pipets
Loops
Air pump
Heater
pH probe
Temperature probe
Fermentor vessel
Autoclave
Ice
Shaker
Ethanol
DH20
Centrifuge
Colorimeter
Balance
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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
Background Information
For over 6000 years, a process known as fermentation has been used for food preservation.
However, it was not until the 1850s that Louis Pasteur demonstrated that microorganisms
were the agents responsible for fermentation. Since his breakthrough, researchers have
learned that fermentation is the result of these microorganisms breaking the chemical bonds
in sugar and starch molecules to create energy. The byproducts of this process, including
lactic acid, ethanol and acetic acid. Some popular fermented products like yogurt, sauerkraut
and wine continue to be consumed on a regular basis.
Current technologies have extended the utility of fermentation, which can now be exploited
to manufacture products as diverse as biofuels, biopharmaceuticals and ne chemicals.
Today, studies in fermentation continue to yield new and exciting advances. For example, mi-
crobial geneticists have identied new strains of microorganisms that grow faster and gener-
ate a wide variety of product metabolites like vitamins and antibiotics. Genetic engineering
and recombinant DNA have allow scientists to produce large amounts of important proteins,
essentially converting cells into living factories. Insulin, which is a hormone used to control
diabetes, was the rst medication for human use that was produced by genetic engineer-
ing. New recombinant medicines, such as antibiotics, interferon and blood clotting factor VIII,
have helped save millions of lives and improved the quality of life for millions more.
Today, commercially relevant fermentation products generally fall into one of the ve follow-
ing groups:
1. The microbial cells themselves: e.g., whole cell yeast extracts, bakers yeast, Lactobacil-
lus, E. coli, etc.
2. Enzymes naturally produced by the microbial cells: e.g., amylase, protease, pectinase,
cellulase, lipase, lactase, streptokinase.
4. Recombinant protein expressed by microbial cells: e.g., insulin, interferon, clotting factor
VIII, the Hepatitis B vaccine.
The demand for these products has encouraged development of novel technologies for
genetic engineering, microbial growth, large-scale production of biomolecules and their
subsequent purication.
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EDVO-Kit 305 Fermentation and Bioprocessing of GFP
Fermentors can be used to grow cultures on vastly different scales. While small cultures
(1-10 liters) can be grown, fermentors are especially useful for very large culture volumes (>
1,000 liters). However, a large-scale fermentation reaction cannot be started in such a large
volume. Instead, a series of scaled inoculations are required. Generally, a very small stock
culture (5-10 ml) of cells is grown, which is then used to inoculate a somewhat greater
volume (200 to 1,000 ml) of fresh medium. To keep nutrients evenly distributed, the culture
is agitated by shaking the culture vessel. When these cultures reach log phase growth, they
are, in turn, used to inoculate an even larger volume (10-100 liters) in a seed fermentor. As
its name suggests, the seed culture is then used to seedor serve as the initial source of
cells forthe nal culture, grown in a production fermentor (1,000 to 100,000 liters).
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Fermentation and Bioprocessing of GFP EDVO-Kit 305
There are three main types of systems used for production -- batch, fed- A. Batch
batch or continuous (Figure 3). In batch fermentation, the sterile growth
medium is inoculated and fermentation proceeds without any addition
Concentration
or removal of medium. Limitations to this system include the build up
of toxins and depletion of nutrients, which can slow culture growth. To
counteract nutrient depletion, fed-batch fermentation relies on the addi-
tion of fresh growth medium at different times; however, no growth me-
dium is removed until the end of the process. During continuous fermen-
tation, fresh growth medium is added while the used culture is removed.
Time
This replenishment of nutrients ensures that the culture remains in log
phase, allowing for maximal biomolecule production. B. Fed Batch
Concentration
vested from the culture. This practice is known as bioprocessing. Some-
times, the product molecule can be secreted directly into the medium
by the cells. However, if the molecule is retained intracellularly, the cells
themselves must be disrupted, or ruptured, to liberate the molecule
of interest for recovery. Once the product is available in the medium, it
can be easily separated from the cells or their debris by centrifugation or
Time
ltration. When puried, the product can nally be utilized for commercial
and/or industrial purposes (summarized in Figure 4). C. Continuous
Concentration
In the late 1970s, Green Fluorescent Protein (or GFP) was isolated from
the jellysh Aequorea victoria. This small protein (approximately 27 ki-
lodaltons) possesses the ability to absorb blue light, and then emit green
light in response. This activity is known as uorescence. After scientists
identied the DNA sequence that coded for GFP, researchers were able to
use genetic engineering to introduce uorescent proteins into organisms Time
other than A. victoria, such as E. coli and C. elegans. Scientists studying
GFP identied mutant proteins with particular amino acid substitutions Figure 3:
that changed the behavior of its chromophore, a special part of the pro- Schematic representation of
tein responsible for its light production (Figure 5). Different changes result cell concentration (blue) and
substrate concentration (red)
A) Batch, B) Fed-Batch and
C) Continuous
Stock culture (5-10 ml)
Cells Supernatant
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EDVO-Kit 305 Fermentation and Bioprocessing of GFP
GFP (and its related uorescent proteins) has become an essential tool in cell and mo-
lecular biology. Proteins can be tagged with uorescent proteins using DNA cloning Figure 5:
strategies and expressed in cells. These tags simplify purication, as the GFP-labeled The molecular
protein can be readily identied using U.V. light. The most signicant contribution of structure of the GFP
GFP involves its use as a visualization tool for the study of biological processes within a chromophore.
living cell using uorescent microscopy techniques. For example, by combining regula-
tory DNA sequences with the GFP gene, scientists can observe patterns of when and
where genes are turned on and off, revealing the role those DNA sequences might
normally play in a cell. In addition, by tagging other proteins with GFP, researches
can determine where those proteins can normally be found in the cell. For example,
GFP-tagged HIV virions are used to follow the virus particles as they enter white blood
cells. In the model organism zebrash (Danio rerio), scientists can label blood vessels
with GFP to observe their growth patterns and networks. In this way, GFP and uo-
rescent microscopy have enhanced our understanding of many biological processes by
allowing scientists to watch biological processes in real-time.
Many times, expression of our gene of interest is under the control of an inducible pro-
moter. A promoter is a sequence of DNA that typically occurs just before (upstream)
of the DNA coding sequence (the sequence that species the amino acid sequence
for a protein). This sequence recruits RNA polymerase to the beginning of the coding
sequence, where it will then transcribe the gene.
In order to express our recombinant protein at the optimal growth stage for bioprocess
applications (mid-log phase), scientists have engineered a genetic on/off switch
known as an inducible promoter. These promoters are only active in the presence of a
particular molecule, like arabinose, tetracycline, or isopropyl--D-thiogalactopyranoside
(IPTG).
In this experiment, the host bacterial strain used for protein expression has been
genetically engineered to contain the gene for a special RNA polymerase (T7), which
is under control of the lac promoter. Under normal circumstances, a protein called lac
repressor binds to the lac promoter and blocks transcription. Lac repressor is inacti-
vated in the presence of IPTG, which allows for the expression of T7 polymerase. T7
RNA polymerase recognizes the T7 promoter on the plasmid, selectively transcribing
large quantities of gfp mRNA. gfp mRNA is translated to produce the uorescent GFP
protein.
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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
Experiment Overview
EXPERIMENT OVERVIEW
Over the course of the fermentation, students will monitor the process parameters (tem-
perature, pH and cell concentration) at regular intervals. Cells will be harvested from the
fermentor at regular intervals for purication of GFP protein. Using an Excel spreadsheet,
students will analyze the data to determine the relationships between these param-
eters..
EXPERIMENT OBJECTIVE:
This experiment is designed to introduce the basic concepts of fermentation and bio-
processing through the production of GFP protein in a small-scale fermentor. At the end
of the activity, students will observe and analyze results and will be able transform the
abstract concepts of fermentation and bioprocessing into an enhanced scientic under-
standing.
Analyze Results.
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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP
Laboratory Safety
Although the bacteria used in this experiment are not considered pathogenic, it is good
practice to follow simple safety guidelines in handling and disposal of materials contami-
nated with bacteria.
2. Exercise extreme caution when working in the laboratory equipment used for heating
and melting reagents can be dangerous if used incorrectly.
4. The E. coli bacteria used in this experiment is not considered pathogenic. Although it
is rarely associated with any illness in healthy individuals, it is good practice to follow
simple safety guidelines in handling and disposal of materials contaminated with bacte-
ria.
6. Always wash hands thoroughly with soap and water at the end of each laboratory pe-
riod.
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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
Figure 6
Inoculate the ask with Bactobeads.
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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP
1. After observing the growth in the small ask from Module I, inoculate the
fermentor with the seed culture (Figure 7).
2. Just after inoculation, add the entire amount of Ampicillin-Module II and IPTG
Module II to the fermentor.
3. Place the temperature, heater and pH probes into the vessel as shown in
Figure 8). Using the probes, record the initial pH and temperature. The pH and
the temperature should measure around 7.0 and 37 C, respectively.
Figure 7
4. Record the pH, temperature, and OD every hour in a table in your lab note- Inoculating the fermentor
book (see below).
5. At each time point, remove 10 ml of the medium from the fermentor using
the syringe (Figure 9). Using these cells, measure the optical density (OD) at
600 nm. Save the remaining cells and store them in the refrigerator (4 C) for
the next Module III. (GFP Isolation).
Figure 9
Taking sample from the
fermentor
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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
1. Centrifuge the six samples collected from the fermentation reaction (T=0 to T=5)
for ten minutes at 4000 rpm.
2. Remove the supernatant from each sample and determine the mass of each cell
pellet in grams. Pre-weigh the 50 ml conical microcentrifuge tube before to sub-
strate the weight of the tube to the sample. In your notebook, record the masses
in a table similar to the one below.
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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP
1. Resuspend the pellets from Module II (day two) in 2 ml of GFP Extraction Buffer-
Module III (Component H). Mix the samples in a shaker at room temperature for ten
minutes.
2. Place your microcentrifuge tube containing the GFP cells in the -20 C freezer for 15
minutes, or until frozen. Lay the tube on its side to ensure rapid freezing.
3. After the cells are completely frozen, remove the microcentrifuge tube from the
freezer and place it in a 37 C waterbath to thaw the cells.
4. Repeat steps 2 3 two more times. (Repeated freezing and thawing will lyse the
cells.)
6. At this point, the supernatant should be bright green because it contains the GFP pro-
tein.
Note: If the supernatant is not uorescent and/or the cell pellet is uorescent,
repeat steps 2-5 (freezing/thawing/centrifugation) until the supernatant is
green.
7. Transfer the supernatant into a clean tube. Measure the absorbance at 477 nm using
the spectrophotometer.
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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305
Compile the classroom results in an Excel le. Plot time vs. pH, time vs. OD, time vs. uores-
cence and OD vs. uorescence.
Study Questions
1. Compare and contrast the three types of fermentation. What kind of fermentation was
performed in this experiment?
2. At which step in the experiment do the cells start producing the uorescent protein?
Why?
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EDVO-Kit 305 Fermentation and Bioprocessing of GFP INSTRUCTOR'S GUIDE
Instructor's Guide
OVERVIEW OF INSTRUCTORS PRELAB PREPARATION:
This section outlines the recommended prelab preparations and approximate time requirement to complete each
prelab activity.
Module III: Prepare the microcentrifuge One hour before performing the experiment. 5 min.
Purification of tubes for the students
GFP Protein
Prepare the waterbath One hour before performing the experiment. 5 min.
Prepare the ice for the students Ten min. before performing the experiment. 5 min.
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INSTRUCTOR'S GUIDE Fermentation and Bioprocessing of GFP EDVO-Kit 305
Pre-Lab Preparations
Fermentation experiments contain antibiotics that are used for the selection of trans-
formed bacteria. Students who have allergies to antibiotics such as penicillin, ampicillin,
kanamycin or tetracycline should not participate in this experiment.
1. In a large ask, dissolve the LB Growth Medium-Module I with 250 ml of distilled water.
Mix until completely dissolved.
3. Allow the medium to cool, then add the entire amount of Growth Additive-Module I to
the ask.
1. In the fermentor vessel, dissolve the LB Growth Medium-Module II with 2.5L of distilled
water. Mix until completely dissolved.
3. Divide centrifuge tubes into groups of 5. Each group will receive one set.
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Please refer to the kit
insert for the Answers to
Study Questions