Alpha Complementation

The concept of alpha-complementation is important because it is a quick, easy, 1-step process of determining whether a transformed bacterial colony has plasmid+insert or not  (The tetracycline resistance of pBR322 works, it is just time consuming).

The key to alpha-complementation is the fact that the lac-Z gene product (B-galactosidase) is a tetramer, and each monomer is made of two parts - lacZ-alpha, and lacZ-omega.  Researchers determined that if the alpha fragment was deleted, the omega fragment is non-functional; however, alpha fragment functionality can be restored in-trans via plasmid. Hence, then name alpha-complementation.

What is needed for this to work as a cloning technique is a strain of E. coli that has the deletion of the lac Z-alpha (lacZ DM15 works well as a genotype), and a plasmid with the lacZ-alpha fragment as the scorable marker (such as pBluescript or pCR2.1). If plain plasmid is successfully transformed into a cell, then the cell will express functional B-galactosidase.  However, if the plasmid+insert is transformed into a cell, then it will express non-functional B-galactosidase (the lac Z-alpha will be disrupted with the insert gene product).  Plate the cells out onto selection media based on the selectable marker, IPTG (induces lac repressor to disengage), and X-gal (chromogenic substrate that yields blue product when cleaved by B-galactosidase)  and the white colonies (non-functional B-galactosidase) are the ones with plasmid + insert; the blue ones have plain plasmid.

Another method of accomplishing the same task is to completely delete the lac operon from the chromosome, but introduce lac-Z-omega fragment on an F fertility factor. This accomplishes the same task as above, with a plasmid with lac-Z-alpha scorable marker.