Question

CRISPR LAB

Of the plates that show evidence of the lacZgene having been cut, which plates also show evidence of the DNA cut having been repaired? Note that repairing DNA does not mean repairing the function of a gene.

Answer 1

The question is in context of LacZ screening:

Background: lacZ gene encodes for b-galactosidase protein which cleaves X-gal a colourless analog of lactose. X-gal when cleaved forms a blue product. In its active state it exists as a homotetramer however a mutant β-galactosidase called as the ω-peptide which is derived from the M15 strain of E. coli has its N-terminal residues deleted and is unable to form a tetramer and thus is inactive. This mutant form of protein however may return fully to its active tetrameric state in the presence of an N-terminal fragment of the protein, the α-peptide. In molecular cloning this is used as a screening tool to select recombinants over non recombinants. The host E. coli strain (used for transformation) carries the lacZ deletion mutant (lacZΔM15) and is non functional in the form of ω-peptide , while the plasmids used carry the α-peptide. Neither is functional by itself. However, when the two peptides are expressed together, as when a plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional β-galactosidase enzyme.

Application: The plasmid carrying the α-peptide contains an internal multiple cloning site (MCS) which is used to cut and insert our DNA of interest and thus α-peptide becomes disrupted. This is called as insertional inactivation. On a plate with Xgal the host with recombinant plasmid will show no blue coloured (or they will be white) colonies as α-peptide(lacZ) is disrupted by the insert DNA. However non recombinants will show blue colour because lacZ functions normally.

So the plates having LacZ gene cut will be in case of recombinant ones because of insertional inactivation and the gene will be non functional so the colour will be white. The cloning is a process of cut (by restriction enzymes) and paste (ligation). I think this is what repairing the cut here means i.e simple ligation after MCS digestion and insertion of foreign DNA. so overall the lac z will be non functional and there will be no blue colonies.

However if there is a cut without any insertion and repair occurs, as far as the lacZgene is in frame it will produce bloue colonies on X-gal plate. But if the LacZ gene is non functional the colonies will be still white in colour.