Prokaryotic Cloning Vectors
Plasmids - can carry up to 8 kb -10 kb.
Bacteriophages - Lambda can carry up to 20 kb, M13 up to few kb.
Cosmids - can carry up to 40 kb.
Basic Vector Design
Origin of replication
low copy number - replication tied to cell division
high copy number - replication controlled by accumulation of repressors
Marker for selection of cells that take up vector
If the insert is cloned into BamHI, then Ampicillin used to select for the cells that have taken up the plasmid, since only they will grow on Amp. Clones are then replica plated onto Ampicillin and Tetracycline.
Markers for selection of cells that take up recombinant vector
Clones that grow on Amp but not tet are the recombinants. This is called negative selection since the recombinants don't grown on tet.
Cloning into the LacZ gene which codes for the beta galactosidase enzyme allows for positive selection. The beta-gal protein metabolizes XGAL into a blue product, thus turning non-recombinant containing cells blue. The recombinant inserts interrupt the beta-gal gene function and therefore the cells with the recombinat plasmids grow but are white. This is commonly referred to as blue/white selection.
Components
Origins of replication
compatability, ColE1, F ...
f1 origin from ss bacteriophage used to make ss DNA
Drug resistance genes
Ampicillin
Tetracycline
Indicator genes
Lac Z
Chloramphenicol
Multiple cloning sites
Replacement Sites
Packaging sites
Lambda cos site
f1 origin/packaging site
Transcription Initiation sites
Sp6, T7, T3
Transcription Promoters
Transcription Terminators
Translation Initiation sites
Gene Orientation
If two genes are oriented in the same direction on a plasmid and a strong transcription termination site does not exist between them, then it is possible for the read-through of transcription from one promoter to also activate the expression of the second gene.
