- #1
TytoAlba95
- 132
- 19
I am completely stumped by this question. I can't understand what is meant by complementation.
Here's an excerpt from Primrose:
'The activity of the enzyme b-galactosidase is easily monitored by including in the growth medium the chromogenic substrate 5-bromo- 4-chloro-3-indolyl-b-D-galactoside (Xgal). This compound is colorless but on cleavage releases a blue indolyl derivative. On solid medium, colonies that are expressing active b-galactosidase are blue in color while those without the activity are white in color. This is often referred to as blue/white screening. Since Xgal is not an inducer of b-galactosidase, the non-substrate (gratuitous) inducer isopropyl-b-D-thiogalactoside (IPTG) is also added to the medium.
The phenomenon of a-complementation of b-galactosidase is widely used in molecular genetics. The starting-point for a complementation is the M15 mutant of E. coli. This has a deletion of residues 11–41 in the lacZ gene and shows no b-galactosidase activity. Enzyme activity can be restored to the mutant enzyme in vitro by adding a cyanogen bromide peptide derived from amino acid residues 3–92 (Langley et al. 1975, Langley & Zabin 1976). Complementation can also be shown in vivo. If a plasmid carrying the N-terminal fragment of the lacZ gene encompassing the missing region is introduced into the M15 mutant, then b-galactosidase is produced (how?), as demonstrated by the production of a blue color on medium containing Xgal.
In practice, the plasmid usually carries the lacI gene and the first 146 codons of the lacZ gene, because in the early days of genetic engineering this was a convenient fragment of DNA to manipulate. Since wild-type b-galactosidase has 1021 amino acids, it is encoded by a gene 3.1 kb in length. While a gene of this length is easily manipulated in vitro, there are practical disadvantages to using the whole gene. As will be seen later, it is preferable to keep cloning vectors and their inserts as small as possible. The phenomenon of a-complementation allows genetic engineers to take advantage of the lac system without having to have the entire Z gene on the vector.'
Here's an excerpt from Primrose:
'The activity of the enzyme b-galactosidase is easily monitored by including in the growth medium the chromogenic substrate 5-bromo- 4-chloro-3-indolyl-b-D-galactoside (Xgal). This compound is colorless but on cleavage releases a blue indolyl derivative. On solid medium, colonies that are expressing active b-galactosidase are blue in color while those without the activity are white in color. This is often referred to as blue/white screening. Since Xgal is not an inducer of b-galactosidase, the non-substrate (gratuitous) inducer isopropyl-b-D-thiogalactoside (IPTG) is also added to the medium.
The phenomenon of a-complementation of b-galactosidase is widely used in molecular genetics. The starting-point for a complementation is the M15 mutant of E. coli. This has a deletion of residues 11–41 in the lacZ gene and shows no b-galactosidase activity. Enzyme activity can be restored to the mutant enzyme in vitro by adding a cyanogen bromide peptide derived from amino acid residues 3–92 (Langley et al. 1975, Langley & Zabin 1976). Complementation can also be shown in vivo. If a plasmid carrying the N-terminal fragment of the lacZ gene encompassing the missing region is introduced into the M15 mutant, then b-galactosidase is produced (how?), as demonstrated by the production of a blue color on medium containing Xgal.
In practice, the plasmid usually carries the lacI gene and the first 146 codons of the lacZ gene, because in the early days of genetic engineering this was a convenient fragment of DNA to manipulate. Since wild-type b-galactosidase has 1021 amino acids, it is encoded by a gene 3.1 kb in length. While a gene of this length is easily manipulated in vitro, there are practical disadvantages to using the whole gene. As will be seen later, it is preferable to keep cloning vectors and their inserts as small as possible. The phenomenon of a-complementation allows genetic engineers to take advantage of the lac system without having to have the entire Z gene on the vector.'