Lecture 21- Biotechnology II: Transformation Flashcards
What is molecular cloning?
The act of isolating a gene of DNA and inserting it into a foreign organism so that it can be expressed.
Once the gene has been isolated and amplified (replicated) using PCR, what is the next step?
The gene must be inserted into another organism. This organism is often yeast or bacteria, as they are fast growing simple organisms.
Why can’t the new DNA just be inserted into the foreign organism? How do we get around this?
PCR fragments that are put in bacterium will be degraded by restriction enzymes that cut DNA at restriction sites that they recognize. To get around this, we use a plasmid.
What is a restriction enzyme and what are the two ways in which restriction enzymes can cut DNA at restriction sites?
A restriction enzyme is an enzyme that will recognize a certain sequence of DNA and cut it. It can perform a blunt cut, which cuts straight, or a sticky cut, which cuts with hang overs.
What are the different components that our plasmids must have for cloning?
They must have an origin of replication, cloning sites, a selective marker, and an inducible promoter.
What is the use of having a cloning site in the plasmid that we insert in the bacteria?
The cloning site is a region containing restriction sites. Restriction enzymes will be used to cut open the plasmid, so that the DNA fragment for the gene we want to add can be inserted.
What is the use of having an origin of replication in the plasmid that we insert in the bacteria?
This allows the plasmid to divide when the cell divides so that the gene can be passed on.
What is the use of having a selective marker in the plasmid that we insert in the bacteria?
The selective marker makes the bacteria resistant to antibiotics. The only way the bacteria keeps the plasmid is if it needs it, so we put the bacteria in antibiotics that would kill it if it weren’t for the selective marker.
Having the antibiotic also ensures that all bacteria living in the antibiotic have the plasmid, as they survive.
What is the use of having an inducible promoter in the plasmid that we insert in the bacteria?
The inducible promoter lets us control the activation of the gene in the foreign organism.
What inducible promoter is often used in bacteria?
The lac operon, this means that the gene is turned on when the bacteria is given lactose!
How is the plasmid actually inserted inside the bacteria?
There are three methods, competent cell, heat shock treatment (most common), and electroporation.
Why is transformation useful?
For example, it allowed us to put the gene for insulin in yeast and mass produce insulin to treat diabetes :D.
