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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.

See page 3 for storage instructions.

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

Material Safety Data Sheets can be found on our website: www.edvotek.com

EDVOTEK and The Biotechnology Education Company


are registered trademarks of EDVOTEK, Inc. BactoBead is a trademark
of EDVOTEK, Inc.

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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP

Experiment Components

Component Storage Check ()


A BactoBeads transformed with 4 C Experiment #305 contains
GFP plasmid material for up to 5 lab
groups.
B LB Growth Media - Module I Room Temp.
C LB Growth Media - Module II Room Temp.
D Growth Additive - Module I -20 C Freezer
E Ampicillin - Module I -20 C Freezer
F Ampicillin - Module II -20 C Freezer
G IPTG - Module II -20 C Freezer
H GFP Extraction Buffer - Module III -20 C Freezer All experiment components
are intended for educational
research only. They are not to
REAGENTS & SUPPLIES be used for diagnostic or drug
purposes, nor administered to
or consumed by humans or
Store all components below at room temperature.
animals.

Component Check ()
Microcentrifuge tubes
Syringe
15 ml Centrifuge tubes
Transfer pipets
Loops

Requirements (NOT included in this experiment)

Air pump
Heater
pH probe
Temperature probe
Fermentor vessel
Autoclave
Ice
Shaker
Ethanol
DH20
Centrifuge
Colorimeter
Balance

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3
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.

3. Metabolites naturally produced by the microbial cells:


a. Primary metabolites that perform physiological functions vital for the normal
growth, development, or reproduction of an organism: e.g., ethanol, citric acid,
lysine, vitamins, polysaccharides.
b. Secondary metabolites that are organic compounds produced by an organism, but
are not necessary for its normal growth, development, or reproduction: e.g., antibi-
otic production.

4. Recombinant protein expressed by microbial cells: e.g., insulin, interferon, clotting factor
VIII, the Hepatitis B vaccine.

5. Chemical compounds modied by microbes (bioconversion): e.g., steroid biotransforma-


tion.

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

OVERVIEW OF MICROBIAL GROWTH Microbial Growth Curve

Fermentation requires a special growth medium that Stationary phase


maintains the proper pH and provides cells with oxy-

Number of cells (log)


gen, water, essential minerals and sources of carbon Death
and nitrogen. Because each organism has different phase

physical and chemical requirements for growth, the Log


formulation of the media can vary greatly depending (exponential)
phase
upon the organism and the process. Specic growth
medium allows cells to remain in a condition that
Lag phase
promotes bioprocessing.
Time
Microbial growth does not occur immediately upon
Figure 1: Microbial Growth Curve.
inoculation of the selected nutrient medium. A post-
inoculation period, called the lag phase, allows the
cells to adapt to the new environment by synthesizing factors
necessary for growth and cell division. Once acclimated to
Agitation
the growing conditions, the microbes enter log phase, time System
during which cells grow and division occurs at an exponential System
rate. This is the optimal stage for bioprocess applications, as Medium Monitor
the biological machinery within the cells is primed for protein
expression. Eventually the rate of growth within a culture
slows due to decreased nutrient availability, and an increased Sensor
concentration of toxic compounds causes some cells to die. Air Probe
When the rate of cell death equals the rate of cell growth, the
culture has entered what is referred to as stationary phase.
The culture will persist in stationary phase until the nutrients Thermal
are exhausted or until the toxins in the culture result in cell ly- Reactor Tank Jacket
sis. At this point, the cells begin to die at an exponential rate
(death phase) (Figure 1).
Submerged
Fermentation Vessels (Bioreactors or Fermentors) aerator Effluent

In practice, fermentation requires the careful selection of Figure 2: Example of Bioreactor.


culture conditions to keep cells in a biologically favorable state
that allows for the production of large quantities of product.
Cells are grown in a piece of equipment known as a fermentor (or bioreactor), which is tted
with sensors that continuously monitor the environmental conditions within a culture during
the fermentation process (Figure 2). This information is used to continuously optimize culture
conditions. Some of the factors fermentors can control include temperature, oxygen levels,
pH, antifoaming agents, and the rate and nature of culture mixing.

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

Once fermentation is completed, the desired biomolecules must be har-

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

GREEN FLUORESCENT PROTEIN (GFP)

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)

Shake flask (200-1,000 ml)

Seed fermenter (>5-100 L)


Figure 4:
Harvesting
Schematic for large-scale fermentation process.

Cells Supernatant

Disruption Kill cells Extraction Kill residual cells

Product purification Discard Product purification Discard

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EDVO-Kit 305 Fermentation and Bioprocessing of GFP

in different patterns of absorption and emission of light, allowing scientists to develop O


a rainbow of uorescent proteins. For the discovery and development of GFP and
other uorescent proteins, Osamu Shimomura, Martin Chale and Roger Tsien were N
awarded the Nobel Prize in Chemistry in 2008. N
HO

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.

RECOMBINANT PROTEIN EXPRESSION IN MICROBIAL CELLS

The manufacture of protein products in microbes is an extremely important application


of genetic engineering. Using recombinant DNA technology, scientists copy specic
genes and insert them into a plasmid, which is a small, extrachromosomal piece of
DNA that is propagated by the bacteria. The gene can then be transcribed by RNA
polymerase and translated into protein, after which it is harvested from the cells.

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

In this experiment, a recombinant GFP protein will be expressed by growing a trans-


formed strain of E. coli in a fermentor. Once the cells have entered log phase, students
will turn on GFP production using a special DNA sequence called an inducible promoter.
This promoter allows for the expression of very high levels of GFP protein in the presence
of the small molecule IPTG (isopropyl--D-thiogalactopyranoside).

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.

BRIEF DESCRIPTION OF THE EXPERIMENT:

Grow the inoculum overnight.

Add the incoculum to the fermentor.

Collect Cells (Temp. (C), %Ethanol, pH, OD 600nm).

Collect the samples and weigh the pellet.

Purify the GFP.

Analyze Results.

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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP

Laboratory Safety

IMPORTANT -- READ ME!

Fermentation experiments contain antibiotics to select for transformed bacteria. Students


who have allergies to antibiotics such as penicilllin, ampicillin, kanamycin or tetracycine
should not participate in this experiment.

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.

1. Wear gloves and goggles while working in the laboratory.

2. Exercise extreme caution when working in the laboratory equipment used for heating
and melting reagents can be dangerous if used incorrectly.

3. Do not mouth pipet reagents - use pipet pumps or bulbs.

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.

5. Properly dispose materials after completing the experiment:


a. Wipe down the lab bench with a 10% bleach solution or a laboratory disinfectant.
b. All materials, including pipets, transfer pipets, loops and tubes, that come in contact
with bacteria should be disinfected before disposal in the garbage. Disinfect materi-
als as soon as possible after use in one of the following ways:
Autoclave at 121C for 20 minutes.
Collect all contaminated materials in an autoclavable, disposable bag. Seal the bag
and place it in a metal tray to prevent any possibility of liquid medium or agar from
spilling into the sterilizer chamber.
Soak in 10% bleach solution overnight.
Immerse open tubes and other contaminated materials into a tub containing a 10%
bleach solution. Soak the materials overnight and then discard. Wear gloves and
goggles when working with bleach.

6. Always wash hands thoroughly with soap and water at the end of each laboratory pe-
riod.

7. If you are unsure of something, ASK YOUR INSTRUCTOR!

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Fermentation
Fermentation and
and Bioprocessing
Bioprocessing of
of GFP
GFP EDVO-Kit
EDVO-Kit 305
305

Module I: Day One

PREPARATION OF SEED CULTURE

1. Using sterile technique, add the entire amount of


Bactobeads to the 250 ml of LB Growth medium
(Component B), Ampicillin-Module I (Component E)
to a nal concentration of 100ug/ml, and Growth
Additive-Module I (Component D).

2. Incubate the ask overnight in a shaker at 37C at


200 rpm

3. Monitor the growth until optical density (OD) reaches


at least 1.0.

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

Module II: Day Two

GROWTH OF GFP IN THE FERMENTOR

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).

Time pH Temperature OD 590


0
1
2
3
4 Figure 8
5 Fermentor vessel with probes

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

Module II: Day Two

CENTRIFUGE AND WEIGH THE SAMPLES

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.

Time Mass (g)

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EDVO-Kit 305
EDVO-Kit 305 Fermentation and
Fermentation and Bioprocessing
Bioprocessing of
of GFP
GFP

Module III: Days Three and Four

PURIFICATION OF GFP PROTEIN (BIOPROCESSING)

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.)

5. Centrifuge the tube in a microcentrifuge for 10 minutes at maximum speed.

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

Analyze the Results

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?

3. Why does the pH of the growth medium change during fermentation?

4. What are the most common commercially available fermentation products?

5. What kind of product is GFP? Is intra- or extracellular?

6. What is the source of the uorescence?

7. Upstream bioprocessing involves optimizing the


microbial growth conditions in order to produce GFP Protein (mg/ml)
the maximum amount of product. As a Biopro-
cess Engineer, you have decided to change the Temperature (C) Trial 1 Trial 2 Trial 3
temperature and the IPTG concentration of your 25 1.51 1.46 1.61
fermentation process in order to increase the GFP 30 2.01 1.90 2.22
yield. You performed the following experiments
and collected the following data. 35 1.37 1.01 1.54
37 0.99 0.62 0.87
Using this data, calculate the average GFP produc- 40 0.63 0.51 0.43
tion under each condition and the standard devia-
tion. Plot your results using Excel. Which condi-
tions would you use? GFP Protein (mg/ml)
Concentration IPTG (uM) Trial 1 Trial 2 Trial 3
0 0.79 0.69 0.66
10 0.95 0.85 0.96
50 1.50 1.46 1.57
100 1.80 1.82 1.78
500 1.24 1.25 1.30
1000 0.75 0.80 0.81

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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.

Preparation For: What to do: When: Time Required:

Prepare and autoclave LB Up to one day before performing the 2 hours


Module I: Growth Medium experiment.
Preparation of
Seed Culture Prepare the shaker at 37 C One hour before performing the experiment. 5 min.

Prepare and autoclave LB Up to one day before performing the 2 hours


Growth Medium experiment.
Module II:
Growth of GFP Prepare probes for the One day to 30 min. before performing the
fermentor experiment. 5 min.
in the Fermentor
One day to 30 min. before performing the
Prepare the centrifuge tubes 5 min.
experiment.

Aliquot the GFP Extraction One day before performing


10 min.
Buffer for each group the experiment.

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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15
INSTRUCTOR'S GUIDE Fermentation and Bioprocessing of GFP EDVO-Kit 305

Pre-Lab Preparations

IMPORTANT READ ME!

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.

MODULE I: PREPARATION OF THE SEED CULTURE

1. In a large ask, dissolve the LB Growth Medium-Module I with 250 ml of distilled water.
Mix until completely dissolved.

2. Autoclave at 121 C for 15 minutes or microwave the medium for 10 minutes.

3. Allow the medium to cool, then add the entire amount of Growth Additive-Module I to
the ask.

4. In the lab, prepare the shaker at 37 C.

MODULE II: GROWTH OF GFP PROTEIN IN THE FERMENTOR

1. In the fermentor vessel, dissolve the LB Growth Medium-Module II with 2.5L of distilled
water. Mix until completely dissolved.

2. Autoclave at 121 C for 15 minutes or microwave the medium for 10 minutes.

3. Thaw the Ampicillin-Module II and IPTG-Module II and immediately place on ice.

4. Distribute a set of centrifuge tubes to each group of students.

5. Prepare the probes, centrifuge, and balance for the class.

MODULE III: PURIFICATION OF GFP PROTEIN

1. Aliquot 2 ml of GFP Extraction Buffer for each group.

2. Arrange a 37 C waterbath and ice buckets for the students.

3. Divide centrifuge tubes into groups of 5. Each group will receive one set.

4. Prepare the centrifuge for the class.

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Please refer to the kit
insert for the Answers to
Study Questions