The Bio-Rad pGLO bacterial transformation kit is commonly used to demonstrate this form of genetic exchange, which occurs in bacteria and eukaryotes and which differs fundamentally from transduction and conjugation. DNA is the genetic material in all living cells and ultimately determines much of an organism's phenotype. Among the key experiments which first demonstrated that DNA carries hereditary information were a series of studies by Frederick Griffith on the bacterium Streptococcus pneumoniae Griffith, Griffith showed that a substance from heat-killed virulent bacteria could transform live avirulent ones into organisms that could kill mice.
A commonly used classroom experiment to demonstrate genetic transformation employs the pGLO plasmid and Escherichia coli as the host. A detailed structure of the plasmid is shown in Figure 1. The bp plasmid contains a gene for a green fluorescent protein GFP , which originally came from a bioluminescent jellyfish called Aequorea victoria and which shows a bright fluorescence when exposed to ultraviolet light. Expression of this gene is under the control of a promoter from the arabinose operon, and so transcription occurs in the presence of this sugar Schleif, The pGLO plasmid also contains a gene for ampicillin resistance so that successful transformants can be distinguished from cells that have not taken up the plasmid DNA by their ability to grow on a medium containing this antibiotic.
There are two origins of replication and numerous sites for restriction endonucleases within the plasmid genome. Structure of the pGLO plasmid. Figure 2 shows the regulation of the arabinose genes in more detail Schleif, The araBAD operon encodes three proteins needed for the degradation of L-arabinose.
Binding of RNA polymerase to this promoter is controlled by a positive regulatory protein called AraC, which is encoded by a separate gene araC that has its own promoter site. The catabolite activator protein CAP binds as a dimer to an additional upstream site and facilitates RNA polymerase binding, so transcription of the araBAD genes is now more efficient. By positioning the L-arabinose 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.
In the absence of arabinose, a dimer of the AraC protein binds to sites I 1 and O 2 , forming a loop in the DNA and blocking transcription. The materials for the classroom transformation experiment with the pGLO plasmid using E. In addition to the basic kit, Bio-Rad sells supplementary kits for the purification of the green fluorescent protein by chromatography catalog no. 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. While many general biology, cell biology, biochemistry, and organic chemistry textbooks introduce carbohydrate structure, this topic is often difficult for students to follow. The standard protocol for pGLO transformation of E.
To demonstrate the specificity of the interaction between sugars and the AraC protein, other carbohydrates can be added to the medium instead. An LB suspension of E. 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.
Students can use a variety of sugars as a way of testing the importance of the number of carbons in the chain and the stereochemistry at each position. Additional sugars that might be included in their experiments are D-arabinose, L-glucose, D-fucose, and L-fucose. For Gram-negative organisms like E. Mosher previously described a student exercise using E. I have found that students can explore the phenomenon of carbon catabolite repression using E. The expression of pGLO from the arabinose promoter site in E.
This project can be extended by looking at the effects of other sugars on the fluorescence of the colonies in transformants containing pGLO in the presence of low concentrations of L-arabinose. This aspect of the system can allow an exploration of antibiotic structure and enzyme specificity. A suspension of HB containing the pGLO plasmid is then streaked onto the plates for single colonies and growth is observed after one to two days.
We have found that the transformants can grow in the presence of ampicillin, penicillin G benzyl penicillin , methicillin, and streptomycin.
As a control, students should also test untransformed samples of E. These bacteria are resistant to streptomycin due to a rpsL50 mutation in HB and, surprisingly, also resistant to methicillin.
Conversely, both transformed and untransformed bacteria are inhibited by chloramphenicol and tetracycline because the enzyme has no effect on them. Students can expand on this experiment by using a variety of compounds in the penicillin and cephalosporin family. Foreign DNA can be introduced into the E. Such "autonomously replicating elements" are referred to as " plasmids " or " vectors ".
In order to " stably retain " the plasmid, there needs to be some type of metabolic reason for the E. If the plasmid contains a gene that codes for a protein that protects against antibiotics, then, only cells that have the plasmid will survive in the presence of that antibiotic. Drug resistance can therefore form the basis of a " selectable marker " for the presence of the plasmid in a sample of E. Ampicillin is an antibiotic for gram negative cells such as E.
Other genes that express other proteins can now be introduced into the plasmid, and the host E. Plasmids are typically abbreviated with an acronym that begins with the lower case "p", and the name can provide some information regarding the person that designed the plasmid, or the contents of the plasmid. Promoters are usually indicated with an acronym that begins with an upper case "P".
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.
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. Results of a pGLO bacterial transformation experiment. 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. For more information, visit our partner 3-D Molecular Designs.
The GFP expressed from the pGLO plasmid illustrates the central doctrine of biology, from the transformation of DNA to the expression of a protein to the visualization of a trait. The bacterial proteome contains thousands of proteins, but only the cloned GFP glows! In its native environment, GFP fluoresces in the deep sea jellyfish, Aequorea victoria.
Incredibly, GFP retains its fluorescent properties when cloned and expressed in E. These extensions link two of the most commonly used techniques in biotechnology labs: transformation and protein purification. Purification of a protein depends on a its chemical or physical properties, such as molecular weight, electrical charge, or solubility. GFP can be separated from others by its size using electrophoresis, and it is extremely hydrophobic, which enables its purification using 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 the 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. Students can explore these separation techniques by growing transformed bacteria in liquid culture to grow overnight, then lysing the cells to release their contents.
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