PLASMIDS Dr. E

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

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

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

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

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.

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.