Blue-white screeningAnother method is using blue-white screening to distinguish the bacterial colonies that contained the recombinant DNA which is recombinant vector is this case. Blue-white screening is rapid, convenient and efficient method to differentiate between recombinant and non-recombinant colonies. The well-characterized bacteria such as E.coli which contained lac Z gene is the lac operon which encodes β-galactosidase (β-gal). This expression is induced by lactose or lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). The β-galactosidase enzyme able to break down x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and give an insoluble blue pigment (4-chloro-3-bromo-indigo).
A section from lacZ gene was deleted to develop non-functional β-galactosidase. Then this section of amino acid which have been deleted was encoded which called α-peptide. Scientists introduce multiple cloning site (MCS) into α-peptide and inserted into plasmid which will generate α-complementation cloning vector. MSC also known as polylinker which contains many restriction site that only present once within plasmid. MCS allow molecular biologist insect DNA fragment into it. So, when the fragment of DNA inserted into MCS site within lacZ gene will disrupt α-complementation and no β-galactosidase formed. As stated, β-galactosidase will break down x-gal in plate to give blue pigment. So, if bacteria carry plasmid with the insertion of DNA fragment in MCS (recombinant vector) will not formed blue colonies and white colonies formed. |