Effects of Antibiotics on
Bacterial Growth and
Protein Synthesis:
Instructor’s Manual
I.
Purpose and Concepts Covered .........................................................................1
II.
Inhibition of Bacterial Growth..............................................................................2
III.
IV.
V.
I.
A.
Preparation for the Laboratory.........................................................................2
B.
Protocol ...........................................................................................................3
C.
Expected Results.............................................................................................4
D.
Troubleshooting ...............................................................................................5
Inhibition of Translation .......................................................................................6
A.
Preparation for the Laboratory.........................................................................6
B.
Supplemental Protocol Information .................................................................8
C.
in vitro Transcription/Translation Protocol ........................................................9
D.
Expected Results...........................................................................................11
E.
Troubleshooting .............................................................................................12
F.
Additional Resources.....................................................................................13
!
This instructor’s
manual is available
online only.
This teaching resource is
made available free of
charge by Promega
Corporation. Reproduction
permitted for noncommercial educational purposes
only. Copyright 2007
Promega Corporation. All
rights reserved.
Alternative Protocol for Laboratories Without a Luminometer ......................14
A.
Expected Results of Alternative Protocol ......................................................14
B.
Additional Discussion Questions for Alternative Protocol ..............................15
Supplier and Ordering Information ...................................................................15
Purpose and Concepts Covered
The purpose of this experiment is to demonstrate the differential effects of various
antibiotics on bacterial growth and translation in an in vitro prokaryotic protein expression
system (S30 E. coli extract).
This laboratory exercise provides an opportunity to cover the following topics:
• translation/protein synthesis
• drug screening to identify new bacteriostatic agents
(to apply information learned here to a new concept)
• the relationship between in vitro and in vivo results in experimental design
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II.
Inhibition of Bacterial Growth
Materials Required
Mueller Hinton broth* (dehydrated medium for reconsitution with water into agar or broth;
BBL Microbiology Cat.# 211443*
Mueller Hinton agar* (BBL Microbiology Cat.# 211438)
Distilled water
Petri dishes (Fisher Scientific Cat.# 08-757-9B)
E. coli cultures with an optical density at 600nm in the range of 0.08 to 0.1 for each
student or group of students
Antibiotic disks (see supplier and ordering information at back of manual)
Small beaker with alcohol and forceps
Sterile cottom swabs
Bunsen burner
Metric ruler
Markers to label plates
*Mueller Hinton medium is standard for this type of test, but LB medium can be substituted.
II.A. Preparation for the Laboratory
1.
Prepare Mueller Hinton broth to grow overnight culture of E. coli. Compositions
are given below.
2.
Prepare Mueller Hinton agar plates. Prepare two plates per student or group of
students.
3.
Prepare cultures of E. coli. Incubate with shaking at 37°C until the culture has an
optical density at 600 nm of 0.08–0.1 or until the turbidity matches that of a
McFarland 0.5 standard, which is used as a turbidity standard to prepare bacterial
cultures. On the day of the lab, dispense the cultures into sterile tubes so that
there is at least 5 ml of culture per student or group of students.
4.
Ensure that each student or group of students has a Bunsen burner, small
beaker with alcohol and forceps, sterile cotten swabs, metric rulers and markers
to label plates.
5.
Prior to the laboratory exercise, equilibrate the Mueller Hinton plates to room
temperature.
Preparation of Agars and Broths
Mueller Hinton broth
Use premixed Mueller Hinton medium, and prepare as instructed by the manufacturer.
Autoclave at 121 °C and 20 psi for 30 minutes. If you do not have access to an autoclave, boil the medium or cook it in a pressure cooker for 1 hour.
Mueller Hinton agar plates
Prepare Mueller Hinton broth as described above but add 15 g of agar per liter.
Autoclave, and allow the medium to cool to approximately 55 °C. Pour into plates.
Store the plates at 4 °C.
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LB broth
Prepare LB broth by combining 5 g yeast extract, 10 g Bacto-tryptone and 5 g NaCl.
Add water to bring the final volume to 1 liter. Mix and autoclave at 121 °C and 20 psi
for 30 minutes.
LB agar plates
Prepare LB broth as described above but add 15 g of agar. Autoclave and allow the
medium to cool to approximately 55 °C. Pour into plates. Store the plates at 4 °C.
II.B. Protocol
!
Do not handle antibiotic disks if you are allergic to the antibiotic.
Day 1
1.
Inoculate one Mueller Hinton agar plate with E. coli. Dip a sterile cotton
swab into the overnight culture of E. coli, and wipe off any excess on the
inside of the tube. Inoculate a Mueller Hinton agar plate by streaking the
entire surface of the plate with the swab, turning the plate 90°, swabbing a
second time, turning the plate 45° and swabbing a third time. Run the swab
around the circumference of the plate. Be sure to cover the entire plate.
Discard the swab.
2.
Repeat Step 1 using a fresh sterile swab to inoculate the second Mueller
Hinton agar plate.
3.
Allow the plates to dry for 5 minutes before placing the antibiotic disks on
the plate surface.
4.
Apply four antibiotic disks to the agar surface of one plate using sterile forceps. To sterilize the forceps, dip them in alcohol, letting the excess drip into
the beaker, and pass them through the Bunsen burner flame. Allow the alcohol to burn off. Be sure to space the disks at least 4–5 cm apart to prevent
overlapping zones of growth inhibition. Press each disk gently with sterile forceps so that the disk makes good contact with the surface of the agar.
Note: Each antibiotic disk will be marked with an abbreviation (Table 1). The
blank disk has no markings.
5.
Repeat Step 4 to apply the other two antibiotic disks and the blank disk to
the second Mueller Hinton agar plate.
6.
Cover the plates. Label the plates with your name, date and organism name.
7.
Invert the plates, and incubate them at 35–37 °C for 18–24 hours.
Day 2
1.
Remove the plates from the incubator. With a metric ruler, measure the
zone of inhibition, the distance between the edge of the disk and edge of
bacterial growth, at the widest point.
Note: Results may be easier to read if plates are placed on a black background or a light box.
2.
Record the results in Table 1. Record whether the compound inhibited
bacterial growth.
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Table 1. Effect of Compounds on Bacterial Growth.
Compound
ampicillin
chloramphenicol
streptomycin
tetracycline
erythromycin
neomycin
blank
Abbreviation
AM
C
S
TE
E
N
Zone of Inhibition
(mm)
Inhibited Bacterial
Growth (Yes or No?)
II.C. Expected Results
Zones will vary based on growth phase of bacteria when plated, depth of agar and
environment. However students should see zones around all of the antibiotics tested
in this laboratory. Table 2 provides guidelines for describing E. coli bacteria as
susceptible, intermediate or resistant.
Table 2. Diameter of Zones of Inhibition for E. coli.
Compound
ampicillin (10 µg)
chloramphenicol (39 µg)
streptomycin (10 µg)
tetracycline (30 µg)
erythromycin (15 µg)
neomycin (30 µg)
blank
Susceptible
14 mm or more
18 mm or more
15 mm or more
19 mm or more
18 mm or more
17 mm or more
NA
Intermediate
12–13 mm
13–17 mm
12–14 mm
11–15 mm
14–17 mm
13–16 mm
NA
Resistant
11 mm or less
12 mm or less
11 mm or less
14 mm or less
13 mm or less
12 mm or less
NA
Discussion
1.
Which compounds were the most effective in inhibiting growth of E. coli
based on the width of the zone of inhibition? Which were the least effective?
2.
Based on the mechanisms of action listed in Table 3, why was growth
inhibited? Can you explain why some of the antibiotics tested were less
effective than others at inhibiting growth of E. coli ? Would you have
expected similar results if you had tested S. aureus in your assay?
Table 3. Modes of Action.
Compound
Mechanism of Action
Ampicillin
Chloramphenicol
Inhibits cell wall synthesis by inhibiting formation of the peptidoglycan
cross-link.
Inhibits prokaryotic peptidyl transferase.
Streptomycin
Inhibits prokaryotic peptide chain initiation, and induces mRNA
misreading.
Tetracycline
Erythromycin
Neomycin
Cycloheximide
Inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small
subunit.
Inhibits prokaryotic peptide chain initiation.
Inhibits prokaryotic translocation through the ribosome large subunit.
Inhibits eukaryotic peptidyl transferase.
Note: This is Table 2 in the Students’ Laboratory Manual.
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II.D. Troubleshooting
Symptoms
No growth on bacterial plates
Colonies appearing within the
lawn of E. coli on the plates
Surface of plate is covered with
water
No zones of inhibition detected
around any disks
Zone of inhibition detected around
blank control disk
Zones of inhibition are widely
varying
Discussion
Plates were too warm or cold when bacteria
were streaked. Be sure to equilibrate plates
to room temperature before use.
Plates were not properly prepared or were
old. Use freshly prepared plates that were
made with unexpired components.
Incubator was not at 37 °C. Double-check
the incubator temperature.
Students streaked plates with a swab that
had been dipped in alcohol. Be sure that
students understand the procedure.
Plate is contaminated with other bacteria
or a fungus. Be sure students follow sterile
techique when plating E. coli and antibiotic
disks.
Plates were too warm or cold when plated,
and condensation covered the agar surface.
Equilibrate plates to room temperature, and
incubate upside down in the incubator.
Condensation on top of plate accumulated
on agar surface during incubation. Incubate
plates upside down in the incubator.
Antibiotic disks were too old. Check
expiration dates. Use fresh disks.
Students dipped disk in alcohol. Make sure
that students understand the procedure.
Make sure that plates are poured to a
uniform depth. In the clinical setting the
standard depth is 4 mm. The depth of the
agar can affect the concentration of the
antibiotic as it diffuses from the disk.
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III. Inhibition of Transcription/Translation
Materials Required for the Laboratory Exercise
E. coli S30 Extract System for Circular DNA (Promega Cat.# L1020)
Steady-Glo® Luciferase Assay System (Promega Cat.# E2510)
pBESTluc™ DNA (1µg/µl)
chloramphenicol (Sigma Aldrich Cat.# C7795
streptomycin sulfate salt (Sigma Aldrich Cat.# S0890)
tetracycline hydrochloride (Sigma Aldrich Cat.# T7660)
erythromycin (Sigma Aldrich Cat.# E5389)
neomycin solution (Sigma Aldrich Cat.# N1142)
cycloheximide ready made solution (Sigma Aldrich Cat.# C4859)
nuclease-free water
pipettes and pipette tips
1.5 ml microcentrifuge tubes
distilled water at room temperature
heat block or water bath at 37 °C
Each E. coli S30 Extract System contains sufficient reagents for 30 in vitro translation
reactions. This exercise requires enough reagents for 10 reactions per student or group
of students. The E. coli S30 Extract System contains enough pBESTluc™ DNA for
20 reactions. Note: Be sure to request an additional vial of the pBESTluc™ DNA from
Promega to ensure that there is enough DNA template for your reactions.
Each Steady-Glo® Luciferase Assay System (Cat.# E2510, 10 ml) includes enough
reagents for 100 luciferase assays. Each student or group of students will be performing
10 luciferase assays. This system is also available in a 100 ml size (Cat.# E2520), which
includes enough reagents for 1,000 assays.
We recommend using the Steady-Glo® Luciferase Assay System to measure light output.
The Luciferase Assay Reagent supplied with the S30 Extract System has a half-life of
10 minutes, whereas the Steady-Glo® Reagent has a half-life of approximately 5 hours,
resulting in less than 13% loss of luminescence per hour. The longer half-life of the
Steady-Glo® Reagent allows students to add the reagent at the bench before proceeding
to the luminometer. If the supplied Luciferase Assay Reagent were used, students would
be required to add the reagent to each tube immediately before measuring luminescence.
III.A. Preparation for the Laboratory
Prepare stock solutions for each compound as directed in Table 4. Prepare the
working solution by diluting the stock solution for each compound to the indicated
concentration in nuclease-free water. Store at the appropriate temperature.
Add 10 µl of nuclease-free water to 10 µl of pBESTluc™ Vector to dilute the vector 1:2
for a final concentration of 0.5 µg/µl. Mix, and dispense the vector into the appropriate
number of aliquots for the number of students or groups of students. Store the
dispensed vector on ice. Store any unused vector at –20 °C.
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Table 4. Preparation and Storage Conditions for Antibiotics.
Sigma
Cat.#
C7795
Recommended
Storage
Temperature
Working Solution
10 mg/ml in water
2–8 °C; use stock
solution within
30 days
10 mg/ml in water
2–8 °C for up to
1 month, or –20 °C
for extended
periods.
Compound Name
Chloramphenicol
Stock Solution
20 mg/ml in ethanol
S0890
Streptomycin
sulfate salt
10 mg/ml in 0.9% NaCl
T7660
Tetracycline
hydrochloride
10 mg/ml in water
10 mg/ml in water
–20 °C
E5389
Erythromycin
20 mg/ml in ethanol
10 mg/ml in water
2–8 °C
N1142
Neomycin solution
10 mg/ml in 0.9% NaCl
10 mg/ml in water
2–8 °C
C4859
Cycloheximide
ready made solution
100 mg/ml in DMSO
10 mg/ml in water
2–8 °C
Up to 4 hours before the lab begins, prepare the Steady-Glo® Reagent by transferring the contents of one bottle of Steady-Glo® Buffer to one bottle of Steady-Glo®
Substrate. Mix by inversion until the substrate is thoroughly dissolved. Store the
reagent at room temperature until use.
Note: Since luciferase activity is temperature-dependent, the temperature of the
Steady-Glo® Reagent should be held constant while quantitating luminescence.
This is achieved most easily by using Steady-Glo® Reagent equilibrated to room
temperature, which is near the temperature optimum of luciferase. To equilibrate the
Steady-Glo® Reagent to room temperature, place the tube containing the Steady-Glo®
Reagent into a container of room-temperature water for 30 minutes prior to use. If
cold reagent is used to detect luciferase activity, luminescence will slowly increase
during the experiments as the reagent warms, introducing inaccuracies. High
temperatures cause an increase in luminescence, but the signal becomes less stable.
This can occur if the reagent is too warm or if the luminometer produces excess heat
within the reading chamber.
Storage Conditions
E. coli S30 Extract System for Circular DNA: Store all components at –70 °C. The
product is sensitive to CO2 (avoid prolonged exposure to CO2 sources such as dry
ice) and multiple freeze-thaw cycles, which may have an adverse affect on activity or
performance. Before use, thaw the components on ice. The S30 extract is temperaturesensitive, so thaw the S30 extract just prior to use.
Steady-Glo® Luciferase Assay System: Store the lyophilized Steady-Glo®
Substrate at –20 °C. The substrate may also be stored at 4 °C for up to one month.
Store the Steady-Glo® Buffer below 25 °C. Storage at room temperature is
recommended to eliminate the need for temperature equilibration when the reagent
is reconstituted. Use the reconstituted Steady-Glo® Reagent on the same day it is
prepared, or store at –20 °C for up to 2 weeks. Storing the prepared reagent at room
temperature will result in a 7% loss of luminescence per 8 hours, 10% loss per
24 hours at 4 °C and 8% loss per 2 weeks at –20 °C. The reagent may be subjected
to up to five freeze-thaw cycles with no effect on potency. Frozen reconstituted
reagent should be thawed below 25 °C to ensure reagent performance. Mix well
after thawing. The most convenient and effective method for thawing or equilibrating
cold reagent is to place it in a water bath at room temperature.
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Test Compounds: Recommended storage conditions for the compounds used in
these experiments can be found in Table 4 and in the literature supplied by Sigma
Aldrich.
Precautions
The lyophilized Steady-Glo® Substrate contains dithiothreitol (DTT) and is therefore
classified as hazardous. The reconstituted reagent is not known to present any
hazards, as the concentration of DTT is less than 1%. However, we recommend the
use of gloves, lab coats and eye protection when working with these or any chemical
reagents. Additional information about potential hazards can be found on the
Material Safety Data Sheet supplied with the reagents.
III.B. Supplemental Protocol Information
This protocol requires pipetting volumes as small as 1.0 µl. Failure to pipet accurately
can result in a shortage of S30 Extract System components or other reagents. The
S30 Extract is the limiting reagent in the S30 E. coli Extract System. The extract can
be viscous, so take care to pipet near the top of the liquid level and do not submerge
the pipet tip to the bottom of the tube, because small droplets of liquid can adhere to
the pipet tip. Consider your students’ levels of experience, and if necessary, provide
the students with a short lesson or reminder on how to pipet accurately.
The reaction may be incubated within a temperature range of 24–37 °C. The fastest
linear rate of protein synthesis occurs at 37 °C for approximately 1 hour, although the
reaction will continue for several hours at a slower rate. Lower temperatures result in
a slower rate of synthesis but often extend the time of the linear rate to several hours.
Protein yields from the S30 Extract System for Circular DNA vary with the template
and conditions used. Typical yields range from 50–250 ng per reaction.
When measuring firefly luciferase activity, background luminescence must be
subtracted from all readings. No background is produced by the Steady-Glo® Reagent
or S30 extract lacking the pBESTluc™ DNA, so background luminescence is a
characteristic of luminometer performance. Some instruments also require verification
of linear response at high light levels (consult the instrument manual). In the laboratory
protocol, students are instructed to measure background by adding 100 µl of
Steady-Glo® Reagent to 100 µl of distilled water. Substract this background reading
from the experimental readings (raw luminescence) to calculate net luminescence.
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III.C. in vitro Transcription/Translation Protocol
Use a fresh pipet tip for each reagent addition.
1.
Prepare enough master mix for 9 in vitro transcription/translation reactions by
combining the following reaction components.
Volume Per
×
Reaction
Component
pBESTluc™ DNA (0.5 µg/µl)
Number of
=
Reactions1
Final
Volume
1.0 µl
Amino Acid Mixture Minus Cysteine,
1mM (mix gently prior to use)
2.5 µl
Amino Acid Mixture Minus Methionine,
1mM (mix gently prior to use)
2.5 µl
S30 Premix Without Amino Acids
(mix gently prior to use)
20 µl
Circular2
S30 Extract,
(mix gently prior to use)
Nuclease-free water
Final volume
15 µl
8.0 µl
50 µl
1You
will assemble 8 reactions in this exercise but will prepare enough master mix for
9 reactions. This should ensure that you have enough master mix.
2The
extract can be viscous, so take care to pipet near the top of the liquid level and do not
submerge the pipet tip to the bottom of the tube, because small droplets of liquid can adhere
to the pipet tip.
2.
Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the
reaction mixture to the bottom of the tube.
3.
Label eight 1.5 ml microcentrifuge tubes as reactions 1 to 8. Pipet 49 µl of the
master mix prepared in Step 1 to each of the tubes.
4.
Pipet 1.0 µl of the compound to a tube containing the master mix as follows:
Compound
ampicillin
chloramphenicol
streptomycin
tetracycline
erythromycin
neomycin
cycloheximide
Tube Number
1
2
3
4
5
6
7
5.
Add 1.0 µl of nuclease-free water to tube 8.
Note: Tube 8 does not contain a compound and will act as the positive control.
This reaction represents the maximum level of protein synthesis.
6.
Vortex each reaction gently to mix. Incubate the reaction at 37 °C for 60 minutes.
7.
Stop the reaction by placing the tubes on ice for 5 minutes.
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Detection of Luciferase Protein
1.
Label nine microcentrifuge tubes 1 to 9. Add 95 µl of distilled water to tubes 1–8.
2.
Add 100 µl of distilled water to tube 9.
3.
Add 5.0 µl of each translation reaction to tubes 1–8.
4.
Add 100 µl of Steady-Glo™ Reagent that has been equilibrated to room
temperature to each tube, and mix by pipetting.
Note: Light intensity is a measure of the amount of luciferase present but also the
enzymatic rate, which depends upon temperature. Be sure that the Steady-Glo®
Reagent has been equilibrated to room temperature (20–25 °C) for reproducible
luciferase assay readings.
5.
Place the reaction in a luminometer or multimode instrument capable of measuring
luminescence. Measure the luminescence and record the results in Table 5.
Consult the appropriate operator's manual for operation of the luminometer, if
necessary.
Note: Luminescence is measured in terms of relative light units (RLU).
Table 5. Effect of Compounds on in vitro Transcription/Translation.
Tube
Number
1
2
3
4
5
6
7
8
Compound
ampicillin
chloramphenicol
streptomycin
tetracycline
erythromycin
neomycin
cycloheximide
no compound
9
no compound; no
translation reaction
Raw Luminescence Net Luminescence
(RLU)
(RLU)
Percent Inhibition of
Protein Synthesis
–
–
Note: This is Table 3 in the Students’ Laboratory Manual.
5.
Calculate the net luminescence by subtracting the background luminescence
measured in tube 9 from the experimental luminescence in tubes 1–8. Record
net luminescence values in Table 5.
6.
Calculate the percent of inhibition for each of the compounds using the equation
given below. Reaction 8 did not contain an inhibitor, and the light output from this
reaction represents the maximum level of protein synthesis.
Net Luminescence in the Presence of Compound
× 100%
Net Luminescence in the Absence of Compound
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III.D. Expected Results
Cycloheximide will not inhibit translation in the E. coli S30 system but does inhibit
translation in eukaryotic systems. Puromycin will efficiently inhibit both eukaryotic and
prokaryotic translation. The other antibiotics tested (chloramphenicol, streptomycin,
tetracycline, erythromycin, and neomycin) only inhibit prokaryotic translation. In
experiments performed at Promega, these compounds showed IC50 values in the
range of 0.1–1.0 ng/µl for the S30 E. coli Extract System. The most potent translational
inhibitors are neomycin, followed closely by streptomycin and erythromycin (Figure 1).
Note: A relative light unit is not an absolute unit of measurement. The number of
relative light units (RLU) produced in a reaction depends on the exact experimental
conditions and can vary significantly, so you should not expect to obtain exactly the
same results as shown in Figure 1. In addition, different luminometer manufacturers
will define a relative light unit differently. Therefore, different luminometers will often
yield different results; the number of relative light units determined with one luminometer may be dramatically higher than that with a different luminometer. This difference
affects all measurements taken with a particular luminometer so that, although the
light output might be dramatically higher, the background will also be proportionally
higher. The net result is that the signal-to-background ratio is consistent.
Chloramphenicol
Tetracycline
Neomycin
Cycloheximide
Streptomycin
Puromycin
Erythromycin
1,000,000
900,000
800,000
Luminescence (RLU)
700,000
600,000
500,000
400,000
300,000
200,000
100,000
0
0.001
0.01
0.1
Antibiotic Concentration (ng/µl)
1
10
6951MA
0
Figure 1. Representative results. All compounds were diluted to 10 mg/ml, then 1:10 serial dilutions
were performed from 1:10 to 1:10,000 (1 mg/ml to 0.001 mg/ml, respectively). One microliter of each
serial dilution was added to a 50 µl in vitro translation reaction containing 0.5 µg of pBESTluc™ DNA.
Reactions were incubated at 37 °C for 1 hour. Luminescence was measured using the Steady-Glo®
Reagent and a Berthold EG&G MicroLumat Plus plate-reading luminometer (RLU factor = 10.0;
integration time = 2.0 seconds).
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Supplemental Discussion Questions
1.
As bacteria acquire resistance to existing antibiotics, new antibiotics need to
be developed to combat antibiotic-resistant bacterial strains. Based on this
protocol, design an experiment to screen for new compounds that preferentially inhibit bacterial translation and could have antibiotic properties.
2.
A population of bacteria exposed to an antibiotic stops growing when the
antibiotic concentration is equal to or higher than the minimum inhibitory
concentration (MIC). How could you design an experiment to determine the
MIC for the compounds tested today?
3.
Some antibiotics are bacteriostatic and inhibit bacterial growth, whereas
other antibiotics are bactericidal and cause bacterial cell death. Based on the
mode of action of the antibiotics used in this lab (Table 2), which antibiotics
would you expect to be bacteriostatic and which bacteriocidal?
4.
What results would you expect if you performed the inhibition of transcription/
translation experiments with a rabbit reticulocyte lysate-base transcription/
translation system rather than the bacterial S30 Extract System? Which
antibiotics would result in cell death, and which antibiotics would have no
effect?
5.
E. coli transformed with the pBESTluc™ Vector will express firefly luciferase
protein. What DNA features are necessary for expression of this protein
from the pBESTluc™ Vector? What RNA features are required?
III.E. Troubleshooting
Symptoms
No light output from the
in vitro transcription/
translation reaction
Low light output from
the in vitro transcription/
translation reaction
Causes and Comments
A component of the in vitro transcription/
translation reaction was omitted. Be sure
that reactions were assembled correctly.
S30 Extract lost activity. The S30 Extract is
temperature-sensitive. Thaw the extract just
before use, and keep on ice while working with
the S30 Extract. Minimize the amount of time
that the extract is spent on ice. Do not subject
the S30 Extract to multiple freeze-thaw cycles.
After the initial thaw, quickly refreeze any
unused S30 Extract in single-use aliquots in a
dry ice-ethanol bath, and store at –70° C.
Impurities in the water used to assemble the
reaction inhibited transcription or translation or
degraded the DNA or RNA molecules in the
reaction. Water purity is extremely important.
If translation efficiencies are low, examine the
quality of the water. Use only nuclease-free
water.
S30 Extract lost activity. The S30 Extract is
temperature-sensitive. Thaw the extract just
before use, and keep on ice while working with
the S30 Extract. Minimize the amount of time
that the extract is spent on ice. Do not subject
the S30 Extract to multiple freeze-thaw cycles.
After the initial thaw, quickly refreeze any
unused S30 Extract in single-use aliquots in a
dry ice-ethanol bath, and store at –70° C.
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III.E. Troubleshooting (continued)
Symptoms
Low light output from
the in vitro transcription/
translation reaction
(continued)
Causes and Comments
The S30 Extract is sensitive to multiple freezethaw cycles. Do not subject the S30 Extract to
multiple freeze-thaw cycles. After the initial
thaw, quickly refreeze any unused S30 Extract
in single-use aliquots in a dry ice-ethanol bath,
and store at –70°C.
Reaction components were not mixed before
use. Be sure that students mix each
component well by pipetting before use.
S30 Extract lost activity. S30 extract is
sensitive to CO2. Do not expose the S30
Extract to CO2 sources such as dry ice for
prolonged periods of time.
Firefly luciferase protein produced in the
transcription/translation reaction lost activity.
Luciferase has an optimal reaction temperature
near room temperature. Temperatures above
30 °C can cause thermal inactivation. Be sure
to equilibrate the Steady-Glo® reagent to
room temperature to avoid thermal inactivation
of the luciferase protein.
Reactions were not assembled correctly. Be
sure that students pipet reaction components
accurately. The S30 Extract can be viscous, so
be sure that students pipet near the top of the
liquid level and do not submerge the pipet tip
to the bottom of the tube, as small droplets of
liquid can adhere to the pipette tip.
III.F. Additional Resources
Additional information about the Steady-Glo® Luciferase Assay System and E. coli
S30 Extract System for Circular DNA can be found at the Promega Web site:
Steady-Glo® Luciferase Assay System: www.promega.com/tbs/tm051/tm051.html
E. coli S30 Extract System for Circular DNA:
www.promega.com/tbs/tb092/tb092.html
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
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IM001
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IV. Alternative Protocol for Laboratories Without a Luminometer
The alternative protocol is suitable for laboratories without a luminometer or multimode
instrument capable of measuring luminescence or for instructors who wish to introduce
students to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The
protocol uses the Transcend™ Non-Radioactive Translation Detection Systems (Cat.#
L5070 and L5080) to detect the luciferase protein. Using these systems, biotinylated
lysine residues are incorporated into nascent proteins during translation. This biotinylated
lysine is added to the translation reaction as a precharged ε-labeled biotinylated lysinetRNA complex (Transcend™ tRNA) rather than a free amino acid. After SDS-PAGE and
electroblotting, the biotinylated proteins are visualized by binding streptavidin-alkaline
phosphatase, followed by colorimetric detection. See the Transcend™ Non-Radioactive
Translation Detection Systems Technical Bulletin TB182 for more information. To use the
alternative protocol, substitute 1–2 µl of Transcend™ tRNA for the same volume of
nuclease-free water. Assemble the reactions as directed in the Student Laboratory Manual,
but omit 1–2 µl of nuclease-free water. Add all components except the Transcend™
tRNA, and gently mix the reaction by pipetting while stirring the reaction with the pipette
tip. If necessary, spin briefly in a microcentrifuge to return the sample to the bottom of the
tube. Add the Transcend™ tRNA after mixing, and incubate the reactions as directed.
IV.A. Expected Results of Alternative Protocol
When using this altenative protocol, the synthesized firefly luciferase protein will be
separated by size using SDS-PAGE. The luciferase migrates at 61 kDa. An apparent
internal translation start results in a second major gene product of 48 kDa. This
plasmid also contains the gene for ampicillin resistance (β-lactamase). β-lactamase
may appear as a faint band migrating at 31.5 kDa. See Figure 2 for typical results.
Many eukaryotic genes contain sequences within the coding region that can function
as ribosomal binding sites when they precede a methionine codon. The presence of
such internal sequences can result in internal translation initiation and the synthesis
of potentially undesired truncated proteins in the prokaryotic system. An example of
this can be seen when expressing the firefly luciferase gene in the E. coli S30
Extract System. The firefly luciferase gene contains 14 methionine codons, several
of which are preceded by potential ribosome binding site (RBS) sequences and
produce truncated translation products.
kDa
97.4 –
69.0 –
46.0 –
2
3
luciferase
(61kDa)
luciferase
internal start
(48kDa)
β-lactamase
(31.5kDa)
0692TA07_4A
30.0 –
1
Figure 2. Coupled in vitro transcription/translation of circular DNA templates using the E. coli
S30 Extract System for Circular DNA. Five microliters of each 50 µl reaction mix was loaded in
each lane. Lanes 2 and 3 show protein products synthesized from pBESTluc™ DNA. Full-length
luciferase migrates at 61 kDa. An apparent internal translation start results in a second major gene
product of 48 kDa. β-lactamase migrates at 31.5 kDa. Lane 1 shows the molecular weight markers;
lanes 2 and 3 are duplicate translation reactions.
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IV.B. Additional Discussion Questions for Alternative Protocol
V.
1.
Full-length firefly luciferase protein has a molecular weight of 61 kDa. The firefly
luciferase gene contains 14 methionine codons, several of which are preceded
by potential ribosome binding site (RBS) sequences. Apply this knowledge to
explain the 48 kDa translation product that can be observed after SDS-PAGE.
2.
How are proteins separated by size in an SDS-polyacrylamide gel?
Supplier and Ordering Information
Ordering Information
Product
E. coli S30 Extract System for Circular DNA
Cat.#
L1020
Provides enough control plasmid for 20 student reactions. (Each student group will perform 10 reactions.) Be sure to
request an adiditional vial of pBESTluc™ DNA to ensuret hat there is enough DNA template for your reactions.
Steady-Glo® Luciferase Assay System
E2510
Provides enough reagent for 100 luciferase assays. (Each student group will be performing 10 luciferase assays.)
Transcend™ Non-Radioactive Translation Detection System
L5070
For Laboratory Use. For laboratories that do not have access to a luminometer or multimode instrument that can measure luminescence. Also, instructors who wish to introduce students to SDS-PAGE can use this system.
Nuclease-Free Water
P1193
For Laboratory Use.
Promega Training Support Program: Discounts for Educators in the United
States
To order Promega products for your teaching laboratory (U.S. only) at a significant
educational discount, visit: www.promega.com/us/trainingsupport/default.htm
Materials Required from Vendors Other than Promega
Product
Mueller Hinton Agar
Mueller Hinton Broth (dehydrated powder)
Ampicillin antibiotic disks (10 µg)
Chloramphenicol antibiotic disks (30 µg)
Streptomycin antibiotic disks (10 µg)
Tetracycline antibiotic disks (30 µg)
Erythromycin antibiotic disks (15 µg)
Neomycin antibiotic disks (30 µg)
Blank paper disks
Ampicillin sodium salt
Chloramphenicol
Streptomycin sulfate salt
Tetracycline hydrochloride
Erythromycin
Neomycin solution
Cycloheximide ready made solution
Petri dishes (10 cm)
Vendor
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
BBL Microbiology
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Fisher Scientific
Cat.#
211438
211443
230705
230733
230942
231344
230793
230882
231039
A2084
C7795
S8090
T7660
E5389
N1142
C4859
08-757-9B
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web
site for more information.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
8/07
IM001
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