1. PLASMIDS
Dr. E

2. What is a plasmid? Small double stranded circular DNA
Naturally found in bacteria
It is completely separate form bacterial chromosomal DNA
It independently self replicates
May give bacteria a survival advantage

3. Why are plasmids useful to us?
Plasmids play an integral part in genetic engineering
It allows us to store our gene of interest until needed
It allows us to move our gene of interest at will from a test tube environment to a cell environment or vise versa.
Allows us to amplify our gene of interest

4. Molecular Cloning Making copies of gene of interest
First must insert gene of interest into plasmid
Second introduce plasmid into bacteria
Bacteria acts as a factory for copying
Example-Insulin

5. pTripEx2 Plasmid
3.6 Kb
Multiple cloning site-for insertion of gene of interest
Ori-origin of replication
Selectable marker- ampicillin resistant gene
Color screen-Lac Z-gene

6. Color Screen Lac-Z produces B- galactosidase
Be-galactosidase-enzyme that cleaves Lactose and X- gal
When cleaves X-gal turns colonies blue
Lac-z gene located in MCS
Thus if a gene of interest is inserted it disrupts Lac Z gene-can’t produce enzyme- can’t cleave X-gal-colonies remain white.

7. Cloning Vector Vs. Expression vector
A plasmid is a type of vector because it can transport a gene of interest into a cell.
Cloning vectors only making copies of gene of interest.
Expression vectors express the gene of interest by transcribing gene into mRNA which is then translated into a protein by ribosomes.
Expression vectors require a promotor sequence.