What is pFluoroGreen?
The plasmid that produces the green fluorescent protein contain- ing the GFP gene is pFluoroGreen™. Only bacterial cells that take up the plasmids will survive selection on ampicillin agar plates. When using pFluoroGreen™, green fluorescent colonies will be visible under long U.V. Light.
What is the purpose of the LB amp ARA plate?
LB/amp/ara (Luria Broth + ampicillin + arabinose): on which only transformed E coli grow. They do fluoresce as the arabinose in the medium causes the promoter to switch on the gene for GFP. The E coli starter culture and plasmid DNA have been freeze-dried.
What is the purpose of arabinose?
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. coli live, it binds tightly to AraC.
Why is ampicillin used in bacterial transformation?
Ampicillin inhibits synthesis of the bacterial cell wall (in bacteria like E. coli , found between the inner and outer cell membranes), resulting in bacteria that are very structurally weak. In the hypotonic media in which these cells grow, cells exposed to ampicillin will swell and burst or not grow at all.
What is the function of plasmids in bacteria?
Plasmids have many different functions. They may contain genes that enhance the survival of an organism, either by killing other organisms or by defending the host cell by producing toxins. Some plasmids facilitate the process of replication in bacteria.
Do plasmids replicate?
The plasmid is a small DNA molecule within a chamber that is physically separated from chromosomal DNA and can replicate independently [6].
Can you tell if these bacteria are ampicillin resistant?
No. You cannot tell if the bacteria are ampicillin resistant just by looking at them. The best test would be to take some of the bacteria growing on the LB plate and streak them on an LB/amp plate. If the bacteria are viable on the LB/amp plate, then they are resistant to ampicillin.
What is ampicillin arabinose?
Ampicillin is an antibiotic, normally E. This is because the particular plasmid has a gene that codes for ampicillin resistance. Arabinose is a simple sugar molecule that “turns on” the gene that codes for GFP production.
What foods contain arabinose?
L-Arabinose is a common component in a plant cell wall and is widely distributed in the plant kingdom. It is a main component of cereal hemicellulose, such as corn, wheat, rye, and rice, pectic substances of beet and apple pulps, and some plant gums.
What is the function of ampicillin?
Ampicillin is used to treat certain infections that are caused by bacteria such as meningitis (infection of the membranes that surround the brain and spinal cord); and infections of the throat, sinuses, lungs, reproductive organs, urinary tract, and gastrointestinal tract.
What does ampicillin do to cells?
Ampicillin inhibits bacterial cell-wall synthesis (peptidoglycan cross-linking) by inactivating transpeptidases on the inner surface of the bacterial cell membrane. Thus ampicillin as an antibiotic provides sterility in cell culture. Hope this helps.
How is plasmid pfluorogreen used as a vector?
The plasmid pFluoroGreen was used as a vector in this experiment. What do we mean when we say, “used as a vector”? pFluoroGreen’s insertion allows for the genetic expression and replication of certain traits, it is literally cut into the cell’s DNA.
What is green fluorescent protein ( GFP ) used for?
pGLO Bacterial Transformation and GFP Kits Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence under ultraviolet light. First isolated from the marine jellyfish Aequorea victoria, the gene encoding GFP is used in cellular and molecular biology as a reporter to detect gene expression in transgenic organisms.
How does GFP retain its fluorescent properties when isolated from E coli?
Incredibly, GFP retains its fluorescent properties when cloned and expressed in E. coli and even when isolated from E. coli and separated on polyacrylamide gels or by chromatography. These amazing properties of GFP allow students to visualize the phenotypic properties of a protein and identify the single protein “band” responsible for the trait.
What was the advantage of GFP over fluorescent dyes?
GFP’s main advantage over conventional fluorescent dyes of the time was the fact that it was non-toxic and could be expressed in living cells, enabling the study of dynamic, physiological processes.
