Cutting DNA b Restriction endonucleases (restriction enzymes) sticky endssticky ends blunt endsblunt ends b Nomenclature EcoRIEcoRI E = genus (Escherichia)E = genus (Escherichia) co = species (coli)co = species (coli) R = strainR = strain I = # of enzymeI = # of enzyme

Blunt & Sticky ends

Pasting DNA b Complementary ends (sticky ends) H-bond b Ligase forms phosphodiester bond to seal strands together.

1.Contains an origin of replication, allowing for replication independent of host’s genome. 2.Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3.Contains a multiple cloning site (MCS)MCS 4.Easy to be isolated from the host cell. A plasmid vector for cloning

Plasmid vectors

Expression vector

How to clone DNA b Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest) b Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase b Introduction of cloning vector into cells (transformation by bacterial cells) b Cloning of cells (and foreign genes) b Identification of cell clones carrying the gene of interest

Screening of the clone b The medium in this petri dish contains the antibiotic Kanamycin b The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance b Note the difference in growth

Blue/White Color Screening lacZ insert functional enzymenonfunctional enzyme X-galproductX-galproduct

Selecting Colonies with Recombinant Plasmids