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What is a plasmid?
A circular piece of DNA that can self-replicate.
What is a restriction endonuclease?
A protein that cuts DNA in specific places; used to insert the pGlo genes.
What role does ligase play in molecular biology?
Molecular glue that sticks the gene to both ends of the plasmid to complete the circle.
What is genetic transformation?
Adding a gene into an expression vector (a plasmid designed for gene expression in cells).
What does antibiotic resistance refer to?
Bacteria evolves to resist antibiotics and survive.
What does the bla gene in the pGlo plasmid provide?
Ampicillin resistance.
What is GFP and where does it originate?
Green fluorescent protein that glows; originates from jellyfish.
How does AraC interact with the promoter in the pGlo plasmid?
AraC binds to the promoter (PBAD) and arabinose, allowing RNA polymerase to bind and transcribe the GFP mRNA.
What is Luria broth used for?
It provides bacteria with everything they need to grow (e.g., nutrition).
What is CaCl2 transformation solution used for?
It puts little holes in E. coli cells to allow DNA to flow through; makes cells 'competent'.
What effect does heat shock have on E. coli?
Makes the cell membrane fluid and the holes much larger, allowing DNA to enter more easily.
What is the function of ampicillin?
An antibiotic that kills bacteria.
What does the agar medium provide for bacterial growth?
A solid gel-like surface for bacteria to grow; remains solid at room temperature and liquid when hot.
What is hydrophobic interaction chromatography?
A method that exploits the interaction of hydrophobic residues on proteins with hydrophobic ligands on a chromatographic resin.
How does salt concentration affect GFP's behavior in chromatography?
High salt concentration strengthens hydrophobic interactions, whereas low salt concentration weakens them, leading to GFP's elution.
What is the elution buffer used for in chromatography?
It washes the GFP away from the beads by reforming it into a hydrophilic protein.
What is the role of lysozyme in the purification process?
It lyses (breaks down) the cell membranes of the bacteria.
What is anion exchange used for?
To purify the DNA by attracting negatively charged DNA to a positively charged resin.
What does RNase A do?
It cuts up RNA to prevent it from interfering with plasmid purification.
What is considered sterile technique?
Methods used to prevent contamination, such as using sterile pipettes and disinfecting surfaces.
How do you calculate transformation efficiency?
Divide the number of transformants by the mass of DNA used.
What does the term 'supernatant' refer to?
The liquid that separates from the solid after centrifugation.
What temperature is used for incubating E. coli in the amplification of plasmids?
37 degrees Celsius.
What is the purpose of adding LB broth after heat shock?
To keep the bacteria alive.
What is the outcome of incubating on ice for 10 minutes during transformation?
It slows down the cells, allowing DNA more time to enter before the membrane repairs.
