Insertional inactivation

Insertional inactivation is a technique used in bacterial DNA technology to identify transformant cells which took up recombinant plasmids.Genetically engineered plasmids are used for this purpose because they have specific features useful for the procedure. These vectors typically carry marker genes, which enable detection of those bacterial cells which took up the plasmid – whether this is the ‘native’ (recircularised), or the recombinant plasmid (one that has a fragment of foreign DNA ligated to it). Using selectable marker genes is a more specific method for selection of only those bacterial cells which took up the recombinant plasmid. Prior to ligation of a foreign DNA insert into the plasmid, the vector is cut by restriction enzyme within the region called a polylinker site. This is located within a gene which is used as a selectable marker. Ligation of the DNA insert into the polylinker site consistently causes disruption of the selectable marker gene – the process known as insertional inactivation. The gene is no longer expressed, and can therefore serve for selection of transformed bacterial cells carrying the recombinant plasmid. Figure.1. presents a range of selectable marker genes which can be inactivated during insertional inactivation. The genes would not encode for the synthesis of their products.

SELECTABLE MARKER GENE	PRODUCT OF THE GENE
lacZ/lacZ'	β-galactosidase
uidA	β-glucuronidase
lux	luciferase
bla /amp	β-lactamase
cat	chloramphenicol                          acetyltransferase
gfp	green fluorescent protein           (Aequorea victoria)

Figure.1. Examples of selectable marker genes and their products.

Example of insertional inactivation using pUC18 plasmid vector

pUC18 is a genetically engineered plasmid characterized by specific features. The vector contains a gene for Ampicillin resistance and an insert of the bacterial lacZ gene. A polylinker site with a number of restriction-enzyme sequences is located within the lacZ gene. The product of a functional lacZ gene is an enzyme called β-galactosidase, which breaks down a chromogenic substrate called X-GAL (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Thus, when bacterial cells containing pUC18 plasmids are grown on an agar-based medium with added X-GAL, the substrate is broken down. Bacterial cells containing pUC18 plasmid give rise to blue colonies. Insertion of a fragment of foreign DNA into the polylinker site causes disruption of lacZ gene, which is no longer functional (insertional inactivation). Recombinant pUC18 plasmids can be identified using LacZα_gene as a selectable marker. This is done by growing bacterial cells containing the recombinant plasmid on an agar-based medium containing X-GAL. The disrupted lacZ gene is not expressed, hence, it doesn’t code for production of β-galactosidase. Consistently, the bacterial cells with the recombinant plasmid cannot break down X-GAL, and these give rise to white colonies.