Answer to Question #24163 in Molecular Biology for Sarah

Question

There are two different explanations for insulin production and they are contradictory. One says that the cDNA is inserted into the lac operon after the B galactosidase enzyme gene because they can share a promoter and this is the only way that a gene can be expressed. Without a promoter, a gene cannot be expressed. However, when they talk about identifying the recombinant plasmid the explanation changes. It says that a plasmid with 2 antiobiotic resistance genes is chosen and the insulin gene is placed in the middle of one of the antibiotic resistance genes to disrupt is function causing the other to act as a marker. Therefore the recombinant DNA can be identified when placed in each of the antibiotics. How can the antibiotic resistance method work when the gene is not placed in the lac operon? How can it be expressed without a promoter? Also how did the first method with the lac operon work? How was the recombinant plasmid identified in it?

I am so confused :S

Answer 1

The Lac operon is normally switched off. When this gene is switched on (this happens naturally when lactose is in the environment) it produces protein which cuts lactose into glucose and galactose. The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment. This results in a characteristic blue color in cells containing a functional β-galactosidase. Blue colonies therefore show that they may contain a vector with an uninterrupted gene of β-galactosidase without insertion of insulin gene. In cells containing the plasmid with an insert no functional β-galactosidase may be formed. Therefore cells transformed with plasmid containing an insert form white colonies. So result of a successful insertion can be identified by the white coloration of cells formed from the unsuccessful blue ones.
Antibiotic resistance is often used as marker. The growth of non-transformed cells is easily prevented through culture on media containing the corresponding antibiotic. Antibiotic selection and white-blue selection can be used simultaneously.