What does the pGLO lab show?
With pGLO bacterial transformation, students learn about genetic engineering as they transform a non-virulent laboratory strain of Escherichia coli (E. coli) with the pGLO plasmid. The procedure involves the CaCl2/heat shock method, which is a standard technique used in many research and biomanufacturing laboratories.
Why was the pGLO LB plate included in the experiment?
The purpose of spreading pGLO on the LB plate was to enable bacteria to grow, otherwise the ampicillin would kill them. If the pGLO is not spread, then the cells would be killed by the ampicillin.
What was the purpose of heat shock in the pGLO lab?
The heat shock step facilitates the entry of DNA into the bacterial cells. Recovery Broth is added to the cell suspension, and the bacteria are allowed to recover for 30 minutes at 37°C.
What is the independent variable in the pGLO lab?
Independent variable: the addition of pGLO, arabinose, and ampicilin. Dependent variable: bacterial growth and if it glows. Constants: size of plates, temperature of water bath, temperature of ice bath, measurement unit, amount of pGLO/ampicilin, etc.
Why does pGLO glow in arabinose?
In the pGLO plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. In the presence of arabinose, the GFP gene is turned on, and the bacteria glow brilliant green when exposed to UV light.
Why does pGLO glow?
Why is pGLO transformation important?
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.
Why did you heat shock your E. coli plasmid mixture and then incubate on ice?
The plasmid-cell mixture then is briefly heated to 45–50°C, allowing the DNA to enter the cell through the disrupted membrane. The heated mixture is then placed back on ice to retain the plasmids inside the bacteria.
How does heat shock affect E. coli?
A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50°C Conceivably by Maintaining Cell Envelope Integrity – PMC. The .
How does the pGLO plasmid work?
The pGLO plasmid contains an origin or replication, a selectable marker, and the gene for Green Fluorescent Protein (GFP). The plasmid also contains a gene for the arabinose C protein, which is a protein that regulates expression from the arabinose BAD promoter (PBAD).
Why do you place the tubes on ice pGLO?
Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice? To get the DNA into the bacteria, we have to poke holes in them with the chemical calcium chloride (CaCl2).