University:
Cameron University
Course:
BIOL 3174 | Molecular Biology
Academic year:

2023
Views:

282

Pages:

2
Author:

Allioniy9p

Blue/White screening (pBLU) A central problem of cloning is the identification of a desired clone among countless bacteria, transformed & untransformed. In the simple cloning system we have used so far, we used antibiotic resistance to select the desired clones. This was possible because our desired clones all carried antibiotic resistance genes that untransformed bacteria did not. But what if you need to select for MORE than just antibiotic resistance? In Labs 18-20, we are cloning a PCR-amplified gene into a plasmid vector (pBLU). After ligation and transformation, we are faced with two variables: • • Tranformed vs untransformed bacteria: Bacteria transformed by pBLU are selected from untransformed cells by resistance to ampicillin (pBLU carries the ampR gene). Bacteria carrying empty pBLU vs bacteria carrying pBLU+PCRproduct (the desired clones): All of these cells will be able to grow on ampicillin. To identify desired clones, use Blue/White screening. Blue/White screening: How does it work? pBLU, and other specially designed cloning vectors, make use of the lac operon. pBLU carries the gene for β-galactosidase (also known as lacZ). This enzyme catalyzes the breakdown of lactose as a food source. It can also degrade an artificial substrate called x-gal, which turns BLUE when it is broken down by β-galactosidase. • Colonies that produce β-galactosidase and are fed x-gal will turn BLUE. • Colonies that do NOT produce β-galactosidase remain white in color, even in the presence of xgal. Plasmid vectors designed for blue/white screening have a multicloning site carefully placed early within the coding region of lacZ. Successful ligation of a foreign DNA into this multicloning site interrupts lacZ and abolishes production of functional β-galactosidase. Therefore: Untransformed bacteria: No growth on amp Bacteria transformed with original pBLU: Blue colonies on amp + x-gal Bacteria transformed with pBLU + insert DNA: White colonies on amp + x-gal Note that blue/white screening is not selection (it does not kill the unwanted bacteria); it is screening. In some blue/white screening systems, an additional reagent must be used: IPTG (isopropylthiogalactoside). IPTG is an inducer that de-represses lacZ expression (it turns the gene on). In some cases, without IPTG, not enough β-galactosidase is produced to turn the colony blue even if the lacZ gene is intact.