What is the difference between positive and negative pglo

Arabinose acts as an allosteric regulator of AraC, changing which DNA sites it binds to and how it forms a dimer. Remember that arabinose is the sugar that gets catabolized by the proteins of the AraBAD operon.

When arabinose is added to the environment in which E. The AraC protein lets go of one of its former binding sites and attaches to another. In this diagram, the two copies of AraC with the green "A" arabinose molecules attached sit side by side on the DNA; in fact, they are stuck together as a new kind of dimer. In this position, AraC no longer acts as a repressor. In eukaryotic gene regulation, a protein that attaches near a promoter and assists RNA polymerase is called a transcription factor.

For some reason, that term is less commonly used for bacterial genes, but I think it fits AraC perfectly in this situation. You encountered this protein acting on the lac operon. The AraBAD operon, like the lac operon, encodes proteins for catabolizing an uncommon type of sugar. In both cases, glucose is the preferred energy source, because fewer enzymes are required. When E. For maximum expression of the GFP gene in pGLO, you'll need to culture your bacteria in plates with arabinose and no glucose.

You'll be able to see how strongly glucose affects GFP expression by looking at your pGLO plates grown with and without glucose added to the medium. Size: the pGLO plasmid is bp. This will become relevant when you analyze the protein and DNA on gels. We also have some mutant versions of pGLO in the lab. One of the mutant versions of the plasmid ends up producing a Blue Fluorescent Protein, and another produces no fluorescent protein at all.

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. Sign In. Skip Nav Destination Article Navigation. Close mobile search navigation Article navigation. Volume 81, Issue 1. Previous Article Next Article. Specificity of Carbohydrate—Protein Interactions. Further Extensions. Article Navigation. Research Article January 01 Deutch Charles E. This Site. Google Scholar. The American Biology Teacher 81 1 : 52— Get Permissions. Cite Icon Cite. Figure 1. View large Download slide. Figure 2.

Asif, A. Revisiting the mechanism involved in calcium chloride induced bacterial transformation. Look at the table you used for setting up your plates. Each plate is an experimental treatment; some of these are controls. The set of experimental treatments is designed to answer specific questions, and for each question you need to compare a result to a negative control.

Thus, you need to save some transformed colonies. Depending on your plate results, you might want to restreak some bacteria from one of your transformation plates onto a new plate.

Check with your instructor to see whether you should do this today. This will give you additional fresh colonies to use in later labs. Use the technique from starting bacterial cultures. For this lab, you should restreak on a plate with ampicillin to ensure that only cells containing the pGLO plasmid will grow. Arabinose is optional in this plate, but might be helpful; discuss this with the instructor. Once you restreak a plate, put it in the incubator so it can grow.

Do not seal this new plate with tape. In addition to the newly restreaked plate described above, you should save some of your transformation plates to make sure you have colonies for later use.

If your results are good, save plate 1 , which has fluorescent pGLO-transformed colonies. If you don't have good colonies on that plate, you may be able to use another plate that has colonies containing pGLO; this could be any plate with ampicillin.

Also save plate 6. Use these short, instructional videos to enrich lessons about bacteria, bacterial transformation, and the green fluorescent protein GFP. Investigate the functional elements of pGLO bacterial transformation, including heat shock, antibiotic selection, promoters, and satellite colony formation.

You can create and edit multiple shopping carts Edit mode — allows you to edit or modify an existing requisition prior to submitting. You will be able to modify only the cart that you have PunchedOut to, and won't have access to any other carts Inspect mode — when you PunchOut to Bio-Rad from a previously created requisition but without initiating an Edit session, you will be in this mode. My Account. Browse Catalog. Life Science Research Back.

Life Science Research Explore all. 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.