What was used as the transformation solution in the pGLO lab?
The standard protocol for transformation of E. coli HB101 with pGLO uses a single type of transformation solution (50 mM CaCl2, pH 6.1) and a single form of heat shock (50 seconds at 42°C).
What is transformed in pGLO lab?
With the pGLO transformation kit, students use a simple procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria, and GFP causes the jellyfish to fluoresce and glow in the dark.
How is pGLO made?
Structure. pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows: Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)
How is pGLO plasmid made?
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.
What is in the tube labeled pGLO?
Bio-Rad’s unique pGLO plasmid contains the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system that can be used to control expression of the fluorescent protein in transformed cells.
What is the product of the bla gene?
b-lactamase enzyme is the product of the bla gene (genes are typically lower case and italicized) If a plasmid contains the bla gene, it will confer resistance to ampicillin to the host E.
What are the 4 important regions on the pGLO plasmid?
The pGLO plasmid contains the following genes: GFP gene, the bla gene, the Ori (origin of replication), the araC gene, the pBAD promoter, and an area of multiple cloning sites Figure 2.
What type of plate must pGLO be grown on?
If your transformed (+pGLO) cells grow on ampicillin (plate 1 or 2) and your un-transformed (-pGLO) cells don’t grow on amp (plate 4), then you know your transformation worked. The negative control (no amp resistance without pGLO) is essential.
Why was ampicillin added to some of the plates in the transformation lab?
This plasmid contains an ampicillin-resistance gene in addition to the GFP gene. Ampicillin is an antibiotic and works by preventing E. coli from constructing cell walls, thereby killing the bacteria.
Why is cacl2 used in transformation?
The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core.
What does pGLO stand for?
|PGLO||Persian Gay and Lesbian Organization (now Iranian Queer Organization)|
What is pGLO bacterial transformation and GFP kits?
Bio-Rad’s pGLO Bacterial Transformation and GFP Kits offer engaging and unforgettable lab activities in which students engineer bacteria to express the Green Fluorescent Protein (GFP) and glow green under UV light.
What is PGLO and how does it work?
A description of what you’ll be working with, including a brief overview of transformation. pGLO: How the plasmid works. In this lab, you’ll transform E. coli cells with the plasmid pGLO, which contains a gene for green fluorescent protein (GFP). This will allow you to grow bacterial colonies that are bright, fluorescent green.
How can I view protein expression in my pGLO transformation lab?
Take your pGLO transformation lab to the next level and explore the structure and function of the expressed GFP. The bacterial proteome contains thousands of proteins, but only the cloned green fluorescent protein (GFP) glows! Use SDS-PAGE to view the expression of proteins in your pGLO bacteria.
Is pGLO necessary for AMP resistance?
The negative control (no amp resistance without pGLO) is essential. Un-transformed cells might grow on amp if they were already amp resistant before you started, or if the “amp” plates didn’t actually contain the ampicillin they were supposed to have. Look closely at your plate #1 (pGLO, amp, arabinose).