To understand blue/white screening, you need to know a bit about β-galactosidase structure and function. β-galactosidase is naturally occurring E. coli protein that is responsible for cleaving lactose into glucose and galactose, which are then further metabolized by the bacterium. Functional β-galactosidase is a homo-tetramer and all four subunits are synthesized from the lacZ gene. The polypeptide’s N-terminal domain is responsible for formation of the homo-tetramer (reviewed in Matthews 2005).
Deletion of only 10 amino acids from the N-terminal β-galactosidase results in a polypeptide that forms non-functional dimmers instead of functional tetramers (Figure 1). However, β-galactosidase activity can be reconstituted in cells carrying N-terminal deletions by simply providing a plasmid carrying a small piece of DNA encoding the missing amino acids. Even though the polypeptides are synthesized in two pieces instead of one, the pieces will associate in the cell to form fully functional β-galactosidase.
Understanding β-galactosidase function led to creating a new recombinant DNA tool. This tool has two parts, and is used to quickly identify E. coli cells that have taken up recombinant plasmids.
Part 1: An E. coli strains in which the lacZ gene is mutated by removing a small piece of DNA that encodes the first 41 amino acids; the remaining sequence is called the ω-fragment. Bacteria with this mutation (lacZΔM15) produce a truncated β galactosidase that cannot metabolize lactose because it cannot form functional tetramers (Figure 2).
Part 2: A plasmid that is engineered to carry the small DNA sequence (referred to as the α-fragment) that encodes the first 41 amino acids. Generally the α-fragment is further engineered to include a short sequence of DNA called the Multiple Cloning Site (MCS) (Figure 2). The MCS includes several restriction enzyme recognition sites that are only present once in the plasmid. These sites make it easy to insert foreign DNA sequences into the MCS.
When a lacZΔM15 strain is transformed with a plasmid carrying the α-fragment, the peptide produced from the plasmid and the truncated polypeptide produced from the bacteria’s genomic DNA spontaneously associate to produce functionalβ galactosidase (Figure 2).
However, if a DNA fragment is inserted into the plasmid’s MCS, a non-functional α-fragment is produced, either because a frame shift mutation has been introduced or the inserted sequence simply results in a improperly folded α-fragment (Figure 3). Without a functional α-fragment, the bacteria cannot assemble a functional β-galactosidase.
When a lacZΔM15 strain are transformed with a plasmid carrying an uninterrupted α-fragment, they will metabolize lactose (Figure 2). Bacteria transformed with a plasmid that carries an insert in the α-fragment will not metabolize lactose (Figure 3). Thus, based on ability to metabolize lactose, we can determine which E. coli cells are transformed with a plasmid carrying an insert.
Screening bacteria using metabolism abilities is cumbersome because it requires replica plating the transformants. Fortunately, β-galactosidase will also cleave the chemical X gal, producing a blue colour. Thus, when plated on media containing X-gal, E. coli harbouring a plasmid carrying an insert will form white colonies, while E. coli harbouring a plasmid without an insert will be blue (Figures 2 & 3).