Bio-Rad Explorer pGLO Bacterial Transformation and GFP Kits use the pGLO plasmid, which contains the gene encoding green fluorescent protein (GFP), in a hands-on bacterial transformation lab activity. Use these kits to teach the central dogma, gene expression and regulation, bacterial transformation, protein separation, and the biomanufacturing process. This page provides presentations, videos, and case studies to help you prepare and bring these concepts to light.
Bacterial Transformation
View this tutorial on how to perform a pGLO bacterial transformation.
Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence under ultraviolet light. First isolated from the marine jellyfish Aequorea victoria, the gene encoding GFP is used in cellular and molecular biology as a reporter to detect gene expression in transgenic organisms. With Bio-Rad pGLO Bacterial Transformation Kits, students perform bacterial transformation and express GFP in a non-virulent laboratory strain of the model organism Escherichia coli (
Bacterial transformation is a fundamental technique in molecular biology. It allows scientists to introduce foreign DNA into bacterial cells and has revolutionized genetic research and engineering by enabling scientists to manipulate and study genes with precision.
The pGLO Bacterial Transformation Kits use the CaCl2/heat shock method for transformation. A standard technique for transforming bacteria used in research laboratories and the biomanufacturing industry, the protocol can accommodate virtually any classroom laboratory. It can be completed with no additional laboratory equipment — insulated cups, warm water, and ice are all that is required. Alternatively, students can use lab equipment such as micropipets, water bath or heat blocks, and laboratory incubators, if available.
The elements of the heat shock protocol and how they work are all explained in the PowerPoint files that accompany each kit.
pGLO Plasmid
The pGLO plasmid is designed to enable (1) replication of the plasmid in bacteria, (2) selection of transformants using ampicillin resistance, and (3) regulation of GFP expression. Bacteria transformed with the pGLO plasmid are selected by ampicillin resistance and then induced to express GFP to glow fluorescent green under UV light!
The essential sequences in the pGLO plasmid include the following:
- GFP — jellyfish gene that encodes green fluorescent protein (GFP)
- ori — origin of pGLO plasmid DNA replication (essential for making more copies of the plasmid)
- bla — gene that encodes β-lactamase, an enzyme that breaks down the antibiotic ampicillin; transformants expressing the bla gene can be selected by adding ampicillin in the growth medium
- pBAD promoter — binds AraC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene
- araC — gene that encodes a regulatory protein that binds to the pBAD promoter; only when arabinose binds to the AraC protein is the production of GFP switched on
- Multiple cloning site — a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of a gene of interest
-
Image
pGLO Map & Sequence
Download a PDF containing the pGLO plasmid map and its complete sequence.
-
Image
pGLO Sequence
Download a text (.txt) file of the complete pGLO plasmid sequence.
Gene Regulation
Gene expression is carefully regulated to allow organisms to adapt to environmental conditions and prevent wasteful production of proteins. Regulation often occurs at the level of transcription from DNA into RNA, specifically at the promoter, where RNA polymerase binds the DNA and begins transcription of the gene.
-
- pGLO LB
-
- pGLO LB/amp
-
+ pGLO LB/amp
-
+ pGLO LB/amp/ara
Results of a pGLO bacterial transformation experiment. Controls that were incubated with no plasmid (-pGLO) grow as a lawn in the absence of ampicillin (LB plate) and do not grow at all in the presence of ampicillin (LB/amp). Transformants grown on ampicillin (LB/amp) grow as colonies but do not show GFP fluorescence; those grown in the presence of both amp and ara (LB/amp/ara) do glow green under UV light.
In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons. The bacterial genes encoding the enzymes needed to metabolize the simple sugar arabinose are a perfect example: Three genes that encode the digestive enzymes involved in breaking down arabinose (araB, araA, and araD) are clustered together in arabinose operon 3, and all depend on initiation of transcription from a single promoter, pBAD. Transcription requires the simultaneous presence of RNA polymerase, a DNA-binding protein called AraC, and arabinose.
- When arabinose is absent, the AraC protein binds to the DNA at the binding site for RNA polymerase, preventing transcription of the digestive enzymes
- When arabinose is present, it interacts with AraC, causing AraC to change shape and allowing RNA polymerase to bind the promoter; araB, araA, and araD are then expressed and can do their job to break down arabinose until the arabinose runs out
The pGLO plasmid contains both the promoter (pBAD) and araC gene, but araB, araA, and araD have been replaced by the single gene that codes for GFP.
In the presence of arabinose, the AraC protein promotes the binding of RNA polymerase to the promoter, which causes transcription of the GFP gene into messenger RNA (mRNA), followed by the translation of this mRNA into GFP. This process is called gene expression.
As they produce more and more protein, the cells expressing GFP fluoresce a brilliant green. In the absence of arabinose, however, AraC no longer facilitates the binding of RNA polymerase, and the GFP gene is not expressed, and bacterial colonies have a wild-type (natural) phenotype — white colonies with no fluorescence.
This is an excellent example of the central dogma of molecular biology in action: DNA > RNA > protein > trait.
The pGLO Bacterial Transformation Kits are carefully designed to include controls that facilitate teaching and learning about the regulation of gene expression. For more details, refer to the PowerPoint files that accompany each kit (posted also in this page).
Green Fluorescent Protein (GFP)
View this informative video about GFP from Phospho Biomedical Animation and other videos available in the YouTube pGLO Bacterial Transformation Playlist.
In nature, GFP fluoresces in the deep-sea jellyfish, Aequorea victoria. GFP has a barrel structure surrounding a central alpha helix that contains the fluorophore. It can be used as an example for discussions of protein secondary structure, parallel and anti-parallel beta sheets, and the use of genes and proteins in biotechnology.
GFP does not require enzymes to fluoresce. Its small size (27 kDa) allows it to be fused to other proteins without disrupting their function but to enable cellular localization and tracking. Osamu Shimomura, Martin Chalfie, and Roger Tsien earned a Nobel Prize in 2008 for their development of GFP as a tool that continues to be used in pioneering new discoveries.
Remarkably, GFP retains its fluorescent properties when expressed in
Purification of a protein depends on its chemical or physical properties, such as molecular weight, electrical charge, or solubility. GFP can be separated by size and net charge using gel electrophoresis, and its extreme hydrophobicity enables purification by hydrophobic interaction chromatography (HIC). When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing other bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in a purer form and retaining its glow.
Students can explore these separation techniques by growing transformed bacteria in liquid culture overnight, then lysing the cells to release their contents. The unique fluorescence of GFP allows real-time monitoring of extraction and purification, key processes used in biotechnology to produce and purify designer proteins with commercial or research value.
pGLO Bacterial Transformation & GFP Kits
Bio-Rad’s pGLO Bacterial Transformation and GFP Kits offer engaging and unforgettable student lab activities in which students engineer bacteria to express the Green Fluorescent Protein (GFP) and glow green under UV light. These kits enable hands-on learning about the central dogma, gene expression and regulation, genetic engineering, protein separation, and the biomanufacturing process.
Learn More
PowerPoint Presentations
-
Image
pGLO Bacterial Transformation (PPT 4.4 MB)
Use this student-facing presentation to facilitate use of the classic pGLO Bacterial Transformation Kit.
-
Image
pGLO Bacterial Transformation for General Biology (PPT 22.1 MB)
Use this student-facing presentation to facilitate use of the pGLO Bacterial Transformation Kit for General Biology.
-
Image
pGLO Bacterial Transformation for AP Biology (PPT 41.4 MB)
Use this student-facing presentation to facilitate use of the pGLO Bacterial Transformation Kit for AP Biology.
-
Image
Bring Inquiry Into Your Classroom with the pGLO Plasmid (PPT 9.06 MB)
Learn how to use pGLO bacterial transformation to illustrate the science and engineering practices described in the NGSS framework.
-
Image
GFP Purification — Electrophoresis and Chromatography (PPT 9.63 MB)
Learn how to use SDS-PAGE and/or chromatography to purify glowing GFP from your pGLO transformants.
pGLO Videos & Webinars
-
Image
YouTube pGLO Bacterial Transformation Playlist
A collection of videos and recorded webinars to enhance understanding of bacterial transformation, GFP, gene regulation, and genetic engineering. Includes useful lab preparation videos, too!
-
pGLO Bacterial Transformation Student Activity Video Quick Guide
Prepare students for the lab portion of the follow along with the pGLO Bacterial Transformation Kit and pGLO Transformation and Inquiry Kit for AP Biology.
Case Studies
-
Image
Case Study: A Role for Bacterial Transformation in Controlling Malaria Transmission (PDF 3.3 MB)
A student-facing extension that is also useful for AP Biology exam prep, this case study connects bacterial transformation with the global fight against malaria.
-
Image
Case Study: Hacking the Gut Microbiome (PDF 1.5 MB)
A student-facing extension that is also useful for AP Biology exam prep, this case study connects bacterial transformation with the role of the microbiome in health and disease.
Other pGLO Resources and Ideas
-
Image
The Biotech Universe Poster
Add color to your wall with this free, full-length poster that highlights the pGLO plasmid and flow of biological information from DNA to RNA to protein to trait.
Add to Cart -
Image
pGLO Art
Have your students make artwork using pGLO transformants! These fun pGLO plates were made by students in Valerie May’s AP Biology course at The Woodstock Academy in Woodstock, Connecticut.
-
Image
Microbes in Space
Read about how high school students designed experiments for the International Space Station with
Bio-Rad pGLO Bacterial Transformation Kits.
