Like the lac operon, the AraBAD operon is negatively controlled by a repressor protein. When no arabinose is present, the repressor protein called AraC in this case binds to operator regions in the DNA and blocks transcription of the operon.
In the diagram, the line represents the double-stranded DNA. AraC proteins form a homodimer : two identical proteins bound together. In this situation, AraC is acting as a negative regulator of transcription in other words, a repressor. In other words, when the level of AraC protein builds up in the cell, it turns off the AraC gene, so no more AraC protein is synthesized until the amount of AraC protein decreases.
Therefore, there is always a small amount of AraC protein present. 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. 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". The ori is the origin of repliation for the pGLO plasmid, bla is the gene that codes for b-lactamase, and is the selectable drug-resistant marker for the plasmid, GFP is the GFP gene and araC is the gene coding for the arabinose C protein.
The bla gene includes a promoter at the 5' end of the gene. This is a weak constitutive promoter always "on" at a low-level. The mRNA will be translated to produce low-levels of the b-lactamase protein. 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.
The unique fluorescent property of GFP allows real-time monitoring of extraction and purification, modeling key processes used in biotechnology to produce and purify designer proteins with commercial or research value. 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.
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