ugg butte Biology 305 Laboratory
To understand blue/white screening, you need to know a bit about galactosidase structure and function. 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 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 galactosidase results in a polypeptide that forms non functional dimmers instead of functional tetramers (Figure 1). However, 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 galactosidase.
Figure 1. Assembly of galactosidase protein from wild type LacZ (left) and deletion mutant LacZM15 (right). Deleting only a few amino acids from the N terminus of the protein results in an improperly assembled enzyme (dimer rather than tetramer) that cannot catalyze the breakdown of lactose. Cartoon of galactosidase structure is adapted from Matthews 2005. Click image for a larger view.
Understanding 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 fragment. Bacteria with this mutation (lacZM15) produce a truncated 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 fragment) that encodes the first 41 amino acids. Generally the 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 lacZM15 strain is transformed with a plasmid carrying the fragment, the peptide produced from the plasmid and the truncated polypeptide produced from the bacteria’s genomic DNA spontaneously associate to produce functional galactosidase (Figure 2).
Figure 2. Complementation of galactosidase activity in lacZM15 mutant bacteria. The lacZ gene located on the bacteria’s chromosome is mutated by removal of the nucleotides that code for the first few N terminal amino acids. galactosidase activity is restored (complimented) when the lacZM15 mutant bacteria are transformed with a plasmid that caries the sequence for these first few amino acids. The lacZ fragment is engineered with the proper transcription and translation start signals so that it is constitutively expressed in the bacterial cell. If X gal is provided in the solid media, colonies containing complemented galactosidase will be blue.
Click image for a larger view.
However, if a DNA fragment is inserted into the plasmid’s MCS, a non functional fragment is produced, either because a frame shift mutation has been introduced or the inserted sequence simply results in a improperly folded fragment (Figure 3). Without a functional fragment, the bacteria cannot assemble a functional galactosidase.
When a lacZM15 strain are transformed with a plasmid carrying an uninterrupted fragment, they will metabolize lactose (Figure 2). Bacteria transformed with a plasmid that carries an insert in the 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, 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).