Bioluminescent Genetic Transformation
Field Trip
Background Information
In the early 1970's, Stanley Cohen discovered that some bacteria
contain small, circular pieces of DNA, which are copied and maintained
in the bacteria and which can be transferred to different bacteria.
We call these circular pieces of DNA plasmids. Since Cohen's
original discovery of plasmids, molecular biologists have developed
a variety of techniques to add useful genes to plasmids and to transfer
the DNA into specific bacterial hosts. One common method of transferring
DNA into bacteria is known as a genetic transformation. These
relatively simple techniques of manipulating plasmid DNA and transferring
it into bacteria are the cornerstones of today's advances in molecular
biology.
In the bioluminescence field trip, the students simulate the genetic
transformation of E. coli bacteria with a plasmid containing the
click beetle luciferase gene. Sterile techniques and
equipment will be discussed and used in this lab.
We use E. coli strain JM109, which is not pathogenic to
humans and is commonly used as a host in molecular biology labs.
This transformation method uses E. coli cells that have been chemically
treated to increase their ability to take up the plasmid DNA. After
this chemical treatment, we refer to the bacterial cells as competent
cells. Even after the chemical treatment, only about 1% to 4%
of the cells are competent to take up DNA. A typical transformation
follows the same steps that we use in this lab, but requires longer
incubation times (about 20 minutes on ice and 60 minutes at 37°
C). Because of the low efficiency of this technique and the
relatively long incubation times, we give the students pre-transformed
cells to ensure success.
After combining the DNA and the bacterial cells, the mixture is
incubated on ice to allow the DNA to bind to the outer wall of the
bacteria. In the lab, we allow about 5 minutes for this step,
which usually takes 15-30 minutes. The bacterial cells are then
heat shocked at 42°C for 45 seconds to encourage the bacteria to
take the DNA into the cell. The transformed cells are returned to
the ice and given growth media, LB broth, to help them recover
and start growing. The cells are allowed to recover for only 5 minutes
at room temperature in this step of our procedure.
In addition to the luciferase gene and the necessary control elements,
the plasmid that we use contains the beta-lactamase gene.
When expressed in bacteria, the beta-lactamase protein allows the
bacteria to counteract the normally lethal effects of ampicillin,
which is a potent antibiotic. Students will plate the transformed
bacteria on agar plates containing LB media with
ampicillin. By doing this, they will select only the bacteria
that has been transformed with the plasmid. Bacteria that do not
contain the plasmid DNA will not grow on the ampicillin plates.
In a typical transformation, the cells are allowed to grow for about
1 hour at 37°C to give the bacteria time to express the beta-lactamase
protein so that they will be resistant to the ampicillin in the
agar plates.
We incubate the plated bacteria overnight at 30-31°C or
at room temperature for 2-3 days. The luciferase protein is less
stable at 37°C (98°F), which is the optimal growth temperature for
many laboratory bacterial strains. So that the students can
see the results of the experiment the same day, they will scrape
transformed bacteria from plates that were prepared by a previous
field trip group. (Your students' plates will provide the bacteria
for a future field trip group.)
Bacterial cells expressing the luciferase protein will be added
to a tube containing the luciferase assay reagent (LAR).
LAR is a solution of 1mM luciferin, which is the luciferase
substrate, in 100mM sodium citrate, pH 5.5. The luciferase
reaction also requires the energy of adenosine triphosphate
(ATP), which is provided by the living bacterial cells in this case,
and oxygen, which is incorporated from the atmosphere by
vigorously shaking the tube containing all of the other components.
In this lab the students will use four, naturally occurring, luciferase
mutants, which produce four different colors of bioluminescent
light: green, yellow-green, yellow and orange.
The students will view the tubes in a dark room and should be able
to easily distinguish the four colors.
If you have any questions or would like more information before
you bring your students to the BTCI for the bioluminescence/genetic
transformation field trip, please give us a call. Alternatively,
bring your questions along and we can discuss them during the lab.
We look forward to seeing you and your group on your scheduled field
trip day. Thank you for your interest in BTCI's Biotechnology Field
Trips program.
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