BIEN3910
Experiment 1
Experiment 1: Aseptic Technique and Cell Mass
Determination
Introduction
Since 1971, Paul Berg constructed the first man-made recombinant DNA molecule, recombinant
DNA technology has revolutionized the field of genetics, biotechnology, gene therapy, and genetic
engineering. With the advances of the technology, many recombinant DNA products for human
therapy are produced. Examples include insulin for diabetes, factor VIII for males suffering from
hemophilia A, human growth hormone and etc. In order to manufacture the products to a
marketable scale, it is our role, the engineers, to design large-scale facility and bioprocessing
procedure. It is essential that we understand the underlying fundamentals of recombinant DNA
technology before we could facilitate the industry and therefore the coming six experiments of this
course, is an attempt to give you an overview of the state-of-the-art knowledge in the field.
Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is
digested by Restriction Endonuclease (Experiment 4) and then incorporated into a single
recombinant molecule by DNA Ligase (Experiment 6). To be useful, the recombinant molecule
must be replicated many times to provide material for analysis, sequencing, production, etc.
Producing many identical copies of the same recombinant molecule is called Cloning. Cloning
can be done either in vitro, by a process called the Polymerase Chain Reaction (PCR)
(Experiment 3) or in vivo, done in unicellular prokaryotes like E. coli, unicellular eukaryotes like
yeast and in mammalian cells grown in tissue culture by introducing the recombinant DNA into
the cell by a process called Transformation (Experiment 6). In in vivo cloning, the recombinant
DNA must be taken up by the cell in a form in which it can be replicated and expressed
autonomously. This is achieved by incorporating the DNA into a Plasmid vector. Details of
plasmid vector will be introduced in Experiment 2.
In the coming experiments, you will insert a gene encoding Green Fluorescent Protein (GFP)
into an expression plasmid vector called pRSET-B. With successful construction, the GFP
should be able to express after introducing into bacteria. The bacteria carrying the
constructed plasmid will be fluorescent when excited with incident light at 489nm under a
fluorescent microscope. The overall procedure of these experiments is depicted in Figure 1.1,
refer to the figure whenever you get lost.
It is essential that sterile technique be maintained when working with bacterial or yeast cultures.
Aseptic technique involves a number of precautions to protect both the cultured cells and the
laboratory worker from infection. The laboratory worker must realize that cells handled in the lab
are potentially infectious and should be handled with caution. Protective apparel such as gloves,
lab coats, and eyewear should be worn.
Care should be taken when handling sharp objects such as needles, scissors, scalpel blades, and
glass that could puncture the skin. Sterile disposable plastic supplies may be used to avoid the risk
of broken or splintered glass.
All materials that come into direct contact with cultures must be sterile. Reusable glassware must
be washed, rinsed thoroughly, and then sterilized by autoclaving or by dry heat before reusing.
BIEN3910
Experiment 1
With dry heat, glassware should be heated 90 min to 2 hr at 160C to ensure sterility. Materials
that may be damaged by very high temperatures can be autoclaved 20 min at 120C and 15 psi.
All media, reagents, and other solutions that come into contact with the cultures must also be
sterile.
Forceps, scissors and spreaders used for bacterial or yeast cultures can be rapidly sterilized by
dipping in 70% alcohol and flaming.
Nutrient media used in bacterial or yeast cultures should be checked routinely for contamination.
pEGFP-N1
pRSET-B
EcoRI
BamHI
GFP cut
with BamHI
& EcoRI
pRSET-B cut
with BamHI &
EcoRI
GFP gene
A mp
r
GFP
A mp r
Plasmid
Complementary Base Pairing
Add DNA Ligase
Introduce to E.coli
pRSET-B
A mp r
Transformed Cell
GFP
Recombinant DNA Molecule
Culture bacteria in
medium with Ampicillin
Ampicillin-resistant
colonies from
transformed cell
Petri dish
Figure 1.1 An overview of recombinant DNA technology
Successful construct
become fluorescent
under microscope
BIEN3910
Experiment 1
Experiment
Objective
In this exercise, media preparation, bacterial inoculation and cell mass measurement will be
introduced.
Medium could be either in solid or liquid form. Solid medium is prepared by adding agar into
liquid medium. Agar is a gelatinous material derived from marine algae, its usual melting point is
97-100ºC and it will solidify at about 42ºC.
Materials
1.
2.
3.
4.
5.
6.
7.
8.
LB Agar
LB Broth
Ampicillin 50 mg/ml
Kanamycin 50 mg/ml
E. coli plate
5 ml suspension of E. coli overnight culture
8 test tubes
Deionized (DI) water
Equipment
1. Bunsen Burner
2. Petri Dish
3. Water Bath
4. BioSafety Cabinet
5. Inoculation Loop
6. Incubator
7. Incubator-Shaker
8. Vortex
9. Spreader
10. Beaker with ethanol
BIEN3910
Experiment 1
Procedure
Each student should perform one set of experiment.
A. Preparation of Agar Plate
1. Turn on the bunsen burner.
2. Have a stack of 4 petri dishes ready.
3. Each group obtain a bottle of 300 ml agar medium from the 50ºC water bath (The medium
was autoclaved).
4. Aseptically add 600 l of ampicillin stock solution (50 mg/ml) to the bottle.
5. Stir mix the content in the bottle.
6. Unscrew the cap, flame the bottle neck briefly (Don’t melt the plastic O-ring!).
7. Pour about 25 ml of medium into each plate.
8. Leave the plates on the bench until the medium solidify.
9. Label the plates with your name.
10. Dry the plates in a BioSafety Cabinet for about 30 min.
B. Inoculation of Bacteria in Liquid and Solid Medium
1. Streak Plate
Purpose of plate streaking is for temporary maintenance of cell or isolation of single colonies
from a mixed culture.
START
1. Use one of the plates you prepared.
2. Flame an inoculation loop until red hot.
3. Allow the loop to cool down near a flame.
4. Pick a colony from the plate provided with the inoculation loop.
5. Streak the loop back and forth across the plate.
6. Turn the plate approximately 90º.
7. Flame the loop and allow it to cool down.
8. Streak on the plate again.
9. Repeat step 6-8 twice.
10. Label the plate as “inoculated”
11. Label another plate without inoculation as “clean”
12. Incubate the plates in a 37ºC incubator overnight.
BIEN3910
Experiment 1
2. Liquid Culture
Liquid culture is prepared when large quantity of cells are required, e.g. for DNA preparation
and protein expression.
1. Aliquot three 5 ml liquid medium in sterile culture
tubes.
2. Aseptically add the amount of 10 µl ampicillin and 5 µl
kanamycin in tube 1 and tube 2 with sterile pipet tips
respectively.
3. Flame an inoculation loop until red hot.
4. Allow the loop to cool down near a flame.
5. Pick a colony from the plate provided with the
inoculation loop.
6. Hold the cap in your pinkie finger
7. Flame the tube neck
8. Vigorously flicking the loop in the medium in tube 1.
9. Flame the tube neck again
10. Replace the cap
11. Repeat step 3 to 10 for tube 2.
12. Leave tube 3 uninoculated.
13. Label the tubes with your name.
14. Incubate the tubes in a 37ºC shaker for overnight.
Remarks: Flame the mouth of the tube whenever opening or closing the tube.
Come back on the next day to observe the results.
BIEN3910
Experiment 1
C. Cell Mass Determination
During the growth of a culture in liquid medium, it is often desirable to estimate the cell
concentration in the medium. This is done most easily by spectrophotometry. The number of
photons scattered is proportional to the mass of cells in a sample. Note that it is scattered light that
is being measured not absorbed light. Measurements are usually done at 600 nm since very few
compounds in cell culture media absorb at that wavelength. As is the case with most
spectrophotometric measurements, there is a range in which the measurement is linear with
concentration. It is important to generate a calibration curve to determine that range and to make
sure that all subsequent samples assayed are diluted (if necessary) to fall within the linear range.
To relate the OD reading to an actual number of cells in a sample, a colony assay is performed.
For a colony assay, a cell suspension is serially diluted and a known volume of the diluted
suspension is spread evenly and grown on an agar plate. At an appropriate level of dilution,
individual colonies will form, one from each original bacteria spread on the plate. The colonies
are then counted, and, based on the concentration of the original cell suspension, the dilution ratio,
and the volume spread on the plate, the concentration of cells in the suspension can be determined.
Cell mass determination can be done as a group.
1. Using the test tubes, make the following dilutions of an overnight culture of E. coli. Vortex the
samples to mix before performing the next dilution.
Dilution Scheme:
Sample Volume of Sample (ml)
1
2
3
4
5
6
7
8
9
10
11
12
5.0 of Stock Suspension
1.0 from Sample 1
1.0 from Sample 2
1.0 from Sample 3
1.0 from Sample 4
1.0 from Sample 5
1.0 from Sample 6
0.1 from Sample 7
0.1 from Sample 8
0.1 from Sample 9
0.1 from Sample 10
0.1 from Sample 11
Volume of
Medium (ml)
0.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
0.9
Dilution
Abs
Factor
600nm
1
1:2
1:4
1:8
1:16
1:32
1:64
1:640
1:6400
1:64000
1:640000
1:6400000
Concentration
(cells/ml)
2. Now transfer 1 ml of each serially diluted sample into cuvettes for samples 1-7. Make sure to
vortex the samples before transferring them to prevent settling.
3. Add 1 ml of culture medium into a cuvette and use it as a blank.
4. Put the other cuvettes into the spectrophotometer in order.
5. Set the wavelength to 600nm.
6. Measure the absorbance of sample 1 - 7 and write down your results.
7. Gently resuspend sample 11 remove 50 µl of the diluted culture from the tube.
8. Apply the culture to the surface of the appropriate plates.
9. Sterilize the spreader in the alcohol, drip off the extra alcohol and flame the spreader. Do not
keep the spreader on the burner!
BIEN3910
Experiment 1
10. Once the flame is out, touch the spreader to the lid of the plate to be sure it is cool. Then keep
turning the plate and move the spreader back and forth at the same time to spread the culture
until all the liquid is absorbed.
11. Repeat step 7-10 for 100 µl of sample 11 and 50 µl and 100 µl of sample 12.
12. Incubate in 37ºC incubator overnight.
13. Choose a plate with appropriate dilution, count the number of colonies and calculate the
concentration of the original culture.
14. Once the standard curve of a particular bacterial strain is established, the concentration of the
same type of culture could be determined by measuring the optical density (OD) of the culture
and refer to the standard curve without going through the colony assay steps again.
Remember!! The sample should be diluted to fall within the linear range of the standard
curve.
Come back on the next day to count the colonies.
BIEN3910
Experiment 1
Prelab Questions
1. What should be done before and after opening a bottle/culture tube cap?
2. Bacteria could be inoculated into either solid medium or liquid medium, give one reason
for using each of the method.
3. What equipment is used to determine cell mass in experiment 1?
4. When we make the series of dilutions in cell mass determination experiment, what must be
done before performing the next dilution?
Lab Report Questions
The report should contain all the results/data and show the sample calculations as
appropriate.
1. Describe and comment the observation in part B. What conclusion can you draw?
2. Complete the table in Part C. Plot the OD versus cell concentration to establish a calibration
curve with regression equation.
3. Calculate the concentration of cells associated with an OD600 of 2.75 by using the results from
question 2. If the concentration cannot be found, suggest a way to solve the problem.
4. With the pipette graduation (P1000 pipette: 1 µL; P200 pipette: 0.2 µL), please estimate how the
pipette reading error is propagated during serial dilution according to the procedure in part C
step 1 and complete the table below.
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Propagated % error due to
reading error in pipetting
0
BIEN3910
Experiment 1
5. To reduce the errors and verify the accuracy of the results, a student repeated the experiment
in Part C three times. The results can be found on CANVAS. Average the results and plot the
calibration curve with standard deviation as error bar.