promoter site in front of the gene for the green fluorescent protein in
the pGLO plasmid, GFP will be formed when the bacteria are grown
in the presence of L-arabinose.
The materials for the classroom transformation experiment with the
pGLO plasmid using E. coli HB101 as the host are sold by Bio-Rad Laboratories (Hercules, California, USA; catalog no. 1660003EDU) and are
widely marketed as part of its Explorer program for Advanced Placement (AP) high school students and lower-level university students.
In addition to the basic kit, Bio-Rad sells supplementary kits for the
purification of the green fluorescent protein by chromatography (catalog
no. 1660005EDU) and for its separation from other E. coli proteins by
SDS-polyacrylamide gel electrophoresis (catalog no. 1660013EDU).
The kits have extensive student study guides and work very reliably.
Here, I describe some extensions of the pGLO transformation experiment that can be used to explore aspects of the system in more detail
and to demonstrate other biological concepts.
Specificity of Carbohydrate–Protein
A key feature of the pGLO system is the interaction between the sugar
Catabolite Repression of GFP
L-arabinose and the AraC protein. While many general biology, cell
biology, biochemistry, and organic chemistry textbooks introduce car-
bohydrate structure, this topic is often difficult for students to follow.
The standard protocol for pGLO transformation of E. coli strain
HB101 calls for adding L-arabinose to LB medium at a concentration
of 6 g L− 1 along with ampicillin at a concentration of 100 mg L− 1. To
demonstrate the specificity of the interaction between sugars and the
AraC protein, other carbohydrates can be added to the medium
instead. For simplicity, I have made up standard LB agar plates and
then spread them with 100 µL of a filter-sterilized 10 mg mL− 1 solution
of ampicillin and 100 µL to 200 µL of a filter-sterilized 60 mg mL− 1
solution of a specific sugar. An LB suspension of E. coli HB101
known to contain the pGLO plasmid or the LB suspension from a
standard transformation experiment is then streaked onto the
plates for single colonies. After incubation for one to two days,
the colonies are exposed to the simple UV light provided by Bio-
Rad and the presence or absence of fluorescence is noted. We have
found that while L-arabinose is a good inducer of GFP expres-
sion, D-glucose, D-galactose, D-fructose, and L-rhamnose are not.
Students can use a variety of sugars as a way of testing the impor-
tance of the number of carbons in the chain and the stereochemis-
try at each position. Additional sugars that might be included in their
experiments are D-arabinose, L-glucose, D-fucose, and L-fucose.
In bacteria, the expression of many degradative genes is controlled by
a process called carbon catabolite repression (Brückner & Titgemeyer,
Figure 1. Structure of the pGLO plasmid. The “ori” arrows
show the direction of plasmid replication by DNA polymerases.
The other arrows show the direction of transcription by RNA
polymerase of the genes for AmpR (a β-lactamase), GFP (the
green fluorescent protein), and AraC (the arabinose regulatory
protein) from the adjacent promoter sites.
Figure 2. Organization of the regulatory region adjacent to the
promoter site for the araBAD genes (PBAD). In the absence of
arabinose, a dimer of the AraC protein binds to sites I1 and O2,
forming a loop in the DNA and blocking transcription. In the
presence of L-arabinose, the modified AraC dimer binds to sites I1
and I2, which allows the binding of RNA polymerase and the
catabolite activator protein (CAP) and subsequent transcription.