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. The effects of different antibiotics can also be demonstrated by spreading samples of an E. Bacteria that cannot degrade the antibiotic will show clear zones of inhibition after one to two days. Methylation of host DNA during replication protects it from cleavage by the restriction endonuclease.
Bacterial strains that are used for transformation and cloning experiments have been engineered to prevent cleavage of the plasmid DNA. To demonstrate the importance of this mutation in controlling the efficiency of transformation, students can attempt the transformation process with other E. We have found that wild-type E. On the other hand, other E. Students may want to try the experiment with other E. Most of these extensions are relatively short and easy to do, and so several of them could be done in a single lab session.
However, some instructors may want to combine them into a longer lab project that extends over several periods. The DNA can then be examined by agarose gel electrophoresis and imaged using either ethidium bromide with a UV transilluminator or a blue stain and a white light illuminator.
The DNA can then be cut with various restriction endonucleases and analyzed in more detail. Figure 1 shows the sites of cleavage by many restriction endonucleases. The standard protocol for transformation of E. There have been many investigations and modifications of this process, which involves the formation of competent cells, the uptake of DNA, and the recovery of transformed cells e. The transformation of E.
Because the pGLO system is both accessible and open-ended, it is suitable for student projects at several different levels. Among the other questions that students may want to explore are 1 How can this system be used to demonstrate that DNA is the genetic material? Instructors who have found experiments they like in these kits might want to develop similar extensions to those described here. I thank the students in BIO Genetics Laboratory at Creighton University for trying out many of these extensions of the pGLO transformation experiment in the fall semesters of and I also thank the faculty and staff in the Department of Biology at Creighton University for their hospitality while I was there.
Recipient s will receive an email with a link to 'Transformation of Escherichia coli with the pGLO Plasmid: Going beyond the Kit' and will not need an account to access the content. Sign In or Create an Account. User Tools. Bio-Rad Products Explore all. Support Explore all. Clinical Diagnostics Explore all. Process Separations Explore all.
Food Science Explore all. Bio-Rad Products Back. Life Science Education Explore all. About the Program Explore all. Corporate Explore all. About Bio-Rad Back. About Bio-Rad Explore all. Investor Relations Explore all. Bacterial Transformation With pGLO bacterial transformation, students learn about genetic engineering as they transform a non-virulent laboratory strain of Escherichia coli E. Gene Regulation Gene expression is carefully regulated to allow organisms to adapt to differing conditions and prevent wasteful production of proteins.
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, 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, which serves as a reporter gene.
Green Fluorescent Protein GFP has a barrel structure surrounding a central alpha helix that contains the fluorophore. Secrets of the Rainforest Kit Simulate the drug discovery process in your classroom with a glowing protein. United States. Bacterial Transformation. Gene Regulation. Green Fluorescent Protein. The concepts behind these labs are presented in a set of related pages on this site:. In addition, there are multiple pages for the experimental methods, which you'll find in the menu.
In Bio 6B, you'll work with the plasmid pGLO in a long series of experiments, using multiple techniques of molecular biology. This recombinant plasmid, created by researchers at Bio-Rad, combines a gene for green fluorescent protein GFP , cloned from a jellyfish, with control elements copied from a bacterial operon. The end result is a system that allows for bacterial expression of a eukaryotic gene.
You'll be able to transform E. The pGLO plasmid offers us an experimental system for working with a wide range of concepts and techniques in molecular biology. The GFP absorbs the short-wavelength, high-energy photons from the UV, and re-emits longer-wavelength, lower-energy green photons.
The gene for GFP originally comes from the jellyfish Aequoria victoria. This remarkable protein has turned out to be useful in research, and researchers have tweaked it to make it even more fluorescent. GFP is often used as a reporter gene; by connecting GFP to another gene so both are expressed together, it becomes possible to see where both the GFP and the gene of interest are expressed.
There are thousands of research papers based on this approach. Fluorescent proteins are common in marine invertebrates especially cnidarians , although the reasons for this are not clear. It's possible that fluorescence is important for communication or camouflage in the deep sea.
Green Fluorescent Proteins also provide protection against superoxide radicals by converting the reactive oygen species into less harmful forms. It's not clear whether this is an important factor in the evolution of these proteins. This level of control was achieved by connecting the GFP coding region with a bacterial promoter. If the entire jellyfish GFP gene, including its eukaryotic promoter, had been inserted into the plasmid, the gene wouldn't be expressed in bacteria.
Those enzymes would only be expressed when arabinose was present, similar to the lac operon. This represents a common approach in genetic engineering: an existing promoter is used to regulate a new gene.
When arabinose is added to E. When arabinose is not present, the amount of GFP expression will be close to zero. In addition to the promoter, this operon has multiple sites where regulatory proteins AraC and CRP bind and upregulate or downregulate transcription. Note that there are two different transcription units regions of DNA that get transcribed.
0コメント