One technique to correct genetic defects in bacteria is to introduce the corrected gene back into bacteria utilizing plasmids, a technique known as a-complementation. An additional advantage of a-complementation is that it can be used to screen for recombinant plasmids.
Many cloning vectors in current use encode for the regulatory sequences and N-terminal of b-galactosidase. Within this coding region is a cloning site for the insertion of recombinant DNA. These vectors are transformed into bacteria that encode the carboxy terminus of b-galactosidase. Neither the plasmid nor the bacteria can encode an active enzyme; the two together complement each other, producing active b-galactosidase.
If colonies are grown on plates containing the enzyme substrate 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal), blue colonies are produced. If the plasmids have recombinant DNA cloned into them, then they fail to produce the amino terminus, resulting in the production of white colonies. This blue/white screening allows researchers to rapidly identify colonies with their recombinant DNA.
This kit is designed to teach students how a genetic defect in bacteria can be corrected by transferring in the correct DNA sequence, a method commonly referred to as complementation.
- Teaches bacterial complementation.
- Students utilizes blue/white bacterial screening.
- Involves bacteria transformation and antibiotic selection
Please refer to the Product Protocol for additional equipment needed.