Plasmid cloning vector must have 3 features:
1. Origin of Replication (ORI) sequence which directs the replication of the plasmid within the host.
2. Dominant selectable marker which enables host cells that carry the plasmid to be easily distinguished. Usually selectable marker is gene conferring antibiotic resistance i.e. ampR or camR (chloramphenicol).
3. Unique restriction enzyme cleavage sites for the insertion of the DNA sequences that are to be cloned.
pBR322 - popular cloning vector
Cloning experiment with pBR322
1) Cells that have not been transformed are sensitive to ampicillin and tetracycline
2) Cells that have been transformed with pBR322 but without the insertion of a DNA fragment (transformants) will be sensitive to ampicillin and tetracycline.
3) Cells that contain a recombinant molecule (recombinants) have lost resistance to tetracycline because the DNA fragment was inserted in the middle of the gene cluster conferring resistance to tetracycline tetR.
Selection of recombinant by replica plating onto agar containing antibiotics.
|Ampicillin and tetracycline||No growth||Growth||No Growth|
replica plating has disadvantage of being time-consuming.
Plasmids with lacZ' gene
1. Cells harbouring normal plasmids are ampicillin resistant and able to synthesise b-galactosidase.
2. Cells with recombinant plasmids are ampicillin resistant but are unable to synthesise b-galactosidase.
The two cell types can be distinguished by:
Plating on agar containing ampicillin and isopropyl-thiogalactosidase (IPTG) - induces lac expression and ensures that expression is not repressed - and X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) - substrate for b-galactosidase which is broken down by the enzyme to give a deep blue product.
1. Non-recombinant colonies (ampR, lacZ+) are blue
2. Recombinant colonies (ampR, lac-) are white
Recombinant colonies can be selected without replica plating by colour.
pUC8 and pUC19 more sophisticated and useful vectors than pBR322
1. Open plasmid containing bacteria with lysozyme to break cell open.
2. In comparison with bacterial chromosome plasmids are light as they are smaller and bacterial chromosome is a complex with protein/DNA. Separate by centrifuging. Plasmids are in the supernatant, called cleared lysate.
3. To differentiate between plasmids and remaining fragments of chromosomal DNA CsCl added to lysate and centrifuged. Ethidium bromide is used to stain the DNA. Chromosomal fragments will be linear whilst intact plasmids are supercoiled. Has the disadvantage of also discarding any nicked plasmids as their DNA becomes linearised.
Plasmid DNA in a single bacterial colony can be detected simply by lysing the bacteria and passing the lysate through gel electrophoresis. The bacterial chromosome is large and cannot enter the gel whilst plasmid DNA will move to a position that is characteristic of its molecular weight. It can be identified by staining with ethidium bromide.
Detection of recombinant molecules
Plasmid mixture will contain: