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Exercise 9: Gene Expression
Enable students to observe the experimental process called bacterial transformation,
Demonstrate the relationship between the genetic constitution of an organism and its physical
students to observe the change in phenotype caused by the uptake and expression of a known
plasmid sequence, and
Reinforce the need for sterile technique when working with bacteria.
The blueprint of life itself is found in DNA. But, life wit
hout its supporting
molecules is not possible. DNA
is transcribed into RNA. As RNA the blueprint that was in the DNA can now be read by the translational
machinery to convert this blueprint message into proteins, the language of the cell.
It is the protein
expressed in a cell that determines the physical and biochemical
of a cell. It
is the molecular machinery that transfers the information from DNA to RNA to a protein capable of
performing a function within the cell that
life. This involve
several complex pathways each
of which is subject to regulation. The first step of this cellular pathway is the synthesis of an RNA molecule
from the DNA template. This process is termed transcription. Upon exiting
the nucleus, a mature RNA can be
translated into a polypeptide sequence. This process is known as translation. These pathways happen, with
some modification, in both eukaryotes and prokaryotes.
As much as this process is regulated by the cell it can
be manipulated by researchers in biology, particularly in prokaryotes.
When we talk about DNA what is typically referred to is nuclear DNA, but there are other forms in which DNA
is found, like plasmids.
Plasmids are small, circular DNA molecules that exi
st apart from the chromosome
in most bacterial species
, in the nucleoid region
lasmids are not essential for survival of the host bacteria
when bacteria are placed into certain environments, plasmids could give them that
advantage that allows bacteria to survive and reproduce in these environments. Plasmids can carry
that, when expressed,
help bacteria survive.
some plasmids can have
antibiotics. A bacte
rial cell containing such a plasmid can live and multiply in
the presence of the
Escherichia coli (E. coli)
isolated in many
parts of the world contain plasmids that carry the genetic information for prot
ein products that interfere with
the action of many different antibiotics. In this laboratory, you will introduce a plasmid that contains an
resistance gene into
is the technological application of biological systems or
their derivatives to make/modify
products or processes for a specific use. Biotechnology plays a vital role in health care to manufacture
insulin), antibiotics and vaccines, in agriculture to produce disease resistant crops, and in
ntal preservation to make biodegradable products. Today, the most commonly used form of
. This field involves the direct manipulation of genes
from one organism to another
from one species
to impart a particular
, such as,
pesticide/herbicide resistance, a longer shelf life, and increased nutritional value of
agricultural crops to an organism of interest. Genetic engineering includes techniques such as
. While the latter process creates multiple copies of a desired gene, transformation
, discovered by
alters the genetic code of a cell through the uptake, incorporation and expression of a
foreign gene provided by a “donor”
In order for transformation to be successful, three
conditions are required: 1) a host into which the foreign
DNA can be inserted, 2) a means of delivering the DNA
into the host cells, and 3) a way to identify and select
the transformed cells.
You will use the bacterium
as the host
organism for the current
bacteria that form the normal flora of the gut. In the
benefit their host by producing vitamin K as
preventing other pathogenic bacteria from
residence. However, certain strains of
and result in food poisoning,
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gastrointestinal and urinary tract
infections, neonatal meningitis and pneumonia.
biotechnology because it has only one chromosome
composed of 5 million base pairs (less than 0.2% of the
human genome), a short reproduction time (cell division
every 20 minutes) and a fairly rapid growth rate.
Delivery of foreign DNA into the host ce
ll is mediated
. Commonly used vectors in genetic
include viruses and plasmids. In today’s experiment
you will use a
to transport the gene
of interest into
cells. Because the chances of a successful transform
experimental setup that will allow researchers to
identify transformed cells is crucial. One way to separate
the transformed from non
transformed cells is by “tagging”
the plasmid with a selectable marker. This is done
to the plasmid that confers some type of selective
like antibiotic resistance
. For example,
a plasmid containing a gene for
ampicillin resistance (
can be used
bacterium into an ampicillin resistant
strain. Through the
acquisition of this gene,
become resistant to
ampicillin (an antibiotic similar to penicillin capable of
killing the bacteria) enabling the
bacterial cells to grow in
One plasmid that you will use is called
contains only 2,686 nucleotide pairs (molecular
weight = 2 x 10
). The small size of this plasmid makes it less susceptible to physical damage during handling.
In addition, smaller plasmids generally replicate more efficiently in bacteria and produce larger numbers of
plasmids per cell. Plasmid
contains an ampici
resistance gene that enables
to grow in the
presence of the antibiotic. Bacteria lacking this plasmid, or bacteria that lose the plasmid, will not grow in the
presence of ampicillin. The ampicillin
resistance gene codes for the enzyme beta
which inactivates ampicillin and other
antibiotics in the beta
lactam family of antibiotics
found in the luminescent bacterium
and contains two genes that code for luciferase (the
enzyme that catal
yzes the light
emitting reaction) and several genes that code for enzymes that produce the
luciferins (the substrates for the light
The second plasmid’s, the
plasmid, MW is
approximately 4.5 x 10
In the laboratory, plasmids can b
e introduced into living bacterial cells by a process known as
transformation. When bacteria are placed in a solution of calcium chloride (CaCl
), they acquire the ability to
take in plasmid DNA molecules.
It increases the cell competency, the cell’s ability to pick up a plasmid.
procedure provides a means for preparing large amounts of specific plasmid DNA, since one transformed cell
gives rise to clones that contain exact replicas of the parent plasmi
d DNA molecule. Following growth of the
bacteria in the presence of the antibiotic, the plasmid DNA can be readily isolated from the bacterial culture.
In this exercise, plasmid
and a control plasmid (
) will be introduce
transformation. There are four basic steps to the procedure:
Treat bacterial cells with CaCl
solution in order to enhance the uptake of plasmid DNA. Such
treated cells are said to be “competent.” (This step should be performed by
before or during the laboratory session.)
Incubate the competent cells with plasmid DNA. Select those cells
that have taken up the plasmid DNA by growth on an ampicillin
medium. Examine the cultures in
Preparation of competent cells (These steps were performed by the instructor)
1. Place a vial of CaCl
solution and the tube of
in the ice bath.
2. Using a sterile pipet, transfer
solution to the tube containing
. Tap the
with the tip of your index finger to mix the solution.
. Incubate the cells for
10 minutes on ice. The cells are then called competent because they
up DNA from the medium. If desired, the cells can be stored in the CaCl
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B. Uptake of DNA by competent cells
There are 2 plasmids involved in this experiment each group will only use 1 of the 2 plasmids.
instructor will assign which plasmid your group will use.
1. Label one small Eppendorf
tube “C” (for control plasmid DNA) or one tube “lux” (for plasmid
2. Place both tubes in an ice bath.
3. Using a sterile micropipette, add 5 µL control plasmid to the tube labeled “C”
5 µL plasmid
tube labeled “lux”. Make sure to kee
p all tubes in the ice until instructed otherwise
Gently tap the tube of competent cells with the tip of your index finger to ensure that the cells are in
5. Using a sterile transfer pipet, add
µL of the competent cells to each of the two tubes.
6. Tap each of these tubes with the tip of your index finger to mix these solutions, and store both tubes on ice
for 15 minutes.
During this time, one member of the group should obtain
of competent cells to each tube and label the tubes “NP” (no plasmid).
Every group will have a no plasmid
tube assigned to them.
the tubes to a water bath preheated to 37ºC, and allow them to sit in the bath
Make sure you are able to identify which tubes below to your group before you place them
in the water bath.
8. Use a sterile pipet to add
L nutrient broth to
the control and lux
150 µL of nutrient broth
into the no plasmid tube.
ncubate the tubes at 37
for 45 minutes.
Use this time to make your prediction (Table
1) and answer
C. Selection of cells that have taken up the plasmid by growth on an ampicillin containing medium
Note: There are a total of 6 plates per
trial. Each group will be working with 3 plates.
The instructor will
assign the plates your group will work with.
Each group will obtain 3 agar plates from the instructor. Label the
plates as indicated in Figure 1. Keep in
mind the instructor assigned
your group the
you will be working with.
2. Using a sterile pipet, remove
bacterial suspension from the “C
remove the lid from the “
” plate, and dispense the bacteria onto the agar. Use a
spread the ba
cteria evenly onto the agar surface.
Use of the cell spreader:
a. Dip the cell spreader in ethanol. Pass the spreader across the flame of the ethanol lamp. Make sure
you only pass it through the flame and not keep it in the flame. Once the ethanol has
burned off keep the
spreader still for about 30 seconds. This allows the spreader to cool down before you start spreading the
cells. Once the spreader has cooled use the spreader to evenly distribute the cell suspension over the
entire surface of the plat
e. Return the cell spreader to the ethanol contained
the same procedure until you have plated al the bacteria.
bacterial suspension from the “
” tube to the “
” plate and spread
these cells onto the
surface as described in the previous step.
4. Cells from the
that did not contain plasmids (NP) should be plated onto two plates
NP) as described
in steps 2
5. Replace the lids on the plates, and leave the plates at room temperature until the liq
uid has been
6. Invert the plates and incubate them
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1. Petri Dish Label
Same figure as in the lab manual (Figure 10.3 pp.110)
D. Examine cultures in the dark
Retrieve your group’s plates from the
Open each plate, one by one, to determine if
growth occurred. If growth occurred, note the growth
type (lawn or colonial). Record your results
in Table 2
Allow at least 3 minutes for the eyes to adjust to the dark in a light
om. View your plates and the
plates of your classmates in the dark and then in the light. Record your results in the following table.
Were the results as expected? Explain possible reasons for variations from expected results
mation Efficiency and answer questions.
(Growth or No Growth)
Reason For Expectations
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Figure 9.2. Experimental Predictions
. (a) Lawn Growth, (b) Colonial Growth. Note: The colonial growth exhibited in this figure is
seen in two large patches. Each patch holds hundreds of individual colonies.
Same figure as in the lab manual (Figure 10.6 pp.112)
Based on your predictions, state your
null and alternative hypotheses regarding what you
expect to see if the
bacteria plated on the ampicillin rich medium were successfully transformed.
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What role did the following compounds or steps play in bacterial transformation?
What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have
taken up the plasmid?)
What does the phenotype of the transformed colonies tell you?
What one plate
would you first inspect to conclude that the transformation occurred successfully?
In nature, DNA uptake by different organisms can impart advantages as well as disadvantages to the
When would genetic transformation be advantageous
to a host organism?
When would genetic transformation be maladaptive to a host organism? What consequences
would there be for the host cells?
Can you think of a case where the uptake of foreign DNA would be advantageous for the
disruptive for the surrounding ecosystem?
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Observed Growth Type
Reasoning for observed
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: (Use the left side of the division to calculate the first transformation efficiency for
and use the right for the second set of calculation for the
Transformation efficiency calculations result in very large numbers. It is normally written in scientific
notation. For example, if the calculated transformation efficie
ncy is 1000 ba
g of DNA it should be
reported as 1 x 10
g. Suppose that an efficiency is calculated as 5000 bacteria/
g of DNA.
This would be reported as 5 x 10
The total amount (µg) of plasmid DNA used can be calculated with
the following formula.
µg DNA = concentration (µg/ µL) of DNA x volume of DNA (µL)
Calculate the total volume of cell suspension prepared in the control DNA tube.
Total volume (µL) = amount (µL) of plasmid + amount (µL) of LB
only a portion of the control DNA solution was added to the LB/Amp
plate, you will need to
calculate the fraction of DNA spread onto this plate using the formula below:
Using the values obtained for questions 3
5, determine the actual amount of DNA (µg) present on
Total amount (µg) of DNA = µg of DNA x Fraction of DNA spread
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Calculate transformation efficiency
Repeat questions 3
7 to calculate the transformation efficiency for the
Compare and contrast the number of colonies on each of the following pairs of plates. What does
each pair of results tell you about the experiment?
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Do your results agree with your predictions in Table
What factors could have
affected transformation efficiency?
What transformation efficiency would you expect for all the other treatments?
Why do the cells transformed with
grow in the presence of ampicillin?
Name one enzyme that is produced by cells transformed with plasmid
that is not produced by the
cells transformed by
Remembering that plasmid size will affect the efficiency of transformation, which plate would be
expected to show the few
How would you improve or extend this experiment?