Expression of Green Fluorescent Protein in E. coli. using kits from BioRad.
Mt. Wachusett Community College Biotechnology Program
Photos by Rhonda Doll
We use two kits from BioRad to teach the concepts of bacterial transformation, bacterial
selection and protein purification. The kits make use of green fluorescent protein which enables
us to follow all steps with a simple UV flashlight. Transformed bacteria glow green so the
students are able to pick a transformed colony to expand (Fig. 1). In the next lab, the cells are
spun down out of the culture broth and again the pellet can be observed with the UV light (Fig.
2). To purify the protein, the cells are lysed and the supernatant is passed over a hydrophobic
interaction column. The GFP band can be followed as it moves through the column and finally
elutes into the collection tube (Fig. 3 and 4).
Figure 1. Examples of agar plates with E. coli colonies. The plate on the left has
colonies expressing GFP. The colonies on the right do not express this protein.
Panel A. shows the plates under white (visible) light.
Panel B. shows the same plates viewed with UV light.
A.
B.
Figure 2.
A.
B.
Tubes showing a bacterial pellet. The bacteria have been transformed with a plasmid
that carries the gene for Green Fluorescent Protein (GFP).
A. As seen with white light.
B. The same pellet with UV light.
Figure 3. Purification of the GFP through a Hydrophobic Interaction Column (HIC). The
protein moves through the column in a band that can be seen in this photo (UV light).
Figure 4. As the protein comes off the column, it can be collected. At this point the GFP
has been separated from the majority of bacterial proteins. The clear tube is flow
through from the column wash just before the GFP is eluted (UV light).