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Phlo gel8/14/2023 ![]() ![]() GFP has a barrel structure surrounding a central alpha helix that contains the fluorophore. 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. 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). Results of a pGLO bacterial transformation experiment. This is an excellent example of the central dogma of molecular biology in action: DNA > RNA > protein > trait. 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 process is called gene expression.Īs they produce more and more protein, the cells expressing GFP fluoresce a brilliant green. 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. The pGLO plasmid contains both the promoter (pBAD) and araC gene, but araB, araA, and araD have been replaced by the single gene that codes for GFP, which serves as a reporter gene. ![]() 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.When arabinose is absent, the AraC protein binds to the DNA at the binding site for RNA polymerase, preventing transcription of the digestive enzymes.Transcription requires the simultaneous presence of RNA polymerase, a DNA-binding protein called AraC, and arabinose. 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. The bacterial genes encoding the enzymes needed to metabolize the simple sugar arabinose are a perfect example. These clusters of genes controlled by a single promoter are called operons. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. 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. Gene expression is carefully regulated to allow organisms to adapt to differing conditions and prevent wasteful production of proteins. Multiple cloning site - a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of a gene of interestīacteria transformed with the pGLO plasmid are selected by ampicillin resistance and when induced to express GFP, they glow fluorescent green under UV light!.araC - gene that encodes the regulatory protein that binds to the pBAD promoter only when arabinose binds to the AraC protein is the production of GFP switched on.pBAD promoter - binds AraC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene.bla - gene that encodes β-lactamase, an enzyme that breaks down the antibiotic ampicillin transformants expressing the bla gene can be selected by placing ampicillin in the growth medium. ![]()
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