LacZ gene and Lac promotor- genetecnology´s little helper
β-galactosidase
LacZ gene is E.coli´s gene for LacZ protein; β-galactosidase, also called beta-gal or β-gal, lactase is sub class of β-galactosidase. It is a hydrolase enzyme that catalyzes sertain hydrolyzes of disaccharides into monosaccharides. β-galactosigase hydrolyzes β-glycosidic bond formed a galactose molecules in lactose, lactosylceramides, ganglioside GM1 and many glycoproteins.
Lactose (beta-D-Lactose) is formed from galactose and glucose and hydolyzed by β-galactosidase.
Lac operon
LacZ gene is part of lac operon, that is formed from promotor, operator, LacZ, LacY, LacA and terminator. LacY encodes β-galactoside permease, a membrane protein that pumps lactose into the cell. Lac A encodes β-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides. Only lacZ and lacY seems to be necessary for lactose catabolism. Lac operon is activated with presens of Catabolite activator protein (CAP) and halts in presens of lac repressor. Lac repressor is secreted in absense of lactose and CAP is secreted in absence of glucose. That is when it is reasonable to produse energy from lactose. This dual control mechanism is known as diauxie.
LacZ as a reporter gene
In gene tecnology and molecular biology reproter gene is a gene that can be attach to a regulatory sequense of another gene of intrest. Reporter genes are used to detect cells that includes gene of intrest.
In bacteria a common reporter gene is lacZ gene. Cells are growth on a medium that contains the substrate analog that can be detected.
Lactose analogs
There are many lactose analogs that are useful for work with the lac operon. Usually they are substituted galactosides, wherw the glucose moiety of lactose is replased by another chemical group. Most common are isopropyl-β-D-thio-galactoside (IPTG), X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) phenyl-β-D-galactose (phenyl-Gal) and ortho-Nitrophenyl-β-galactoside (ONPG).
IPTG creates a chemical bond with the sulfur (S), which can´t be hydrolyzated. IPTG binds to the lac repressor and inactivates it. This will trigger transcription of lac operon. IPTG is used in vivo studies, because it doesn´t metabolized by E.coli it´s consentration is constant.
X-gal is consisting of galactose linked to a substituted indole. When β-galactosidase cleaves bond between them and when free indole is oxidized it will turn blue. X-gal it self is colorless.
ONPG is consisting of galactose linked to a substituted ortho-nitrophenol. Free ortho-nitrophenol has a yellow color. ONPG it self is colorless.
Blue white screening
Blue white screening is tecnique that is used to detection of successful ligantions in vector-based gene cloning. When competent cells are growth with X-gal, if bacterial colony will turn white ligantion is succeed, if it turns blue it is´n. This is baced on alpha-complementation. LacZ gene whitch is split in two LacZα and LacZΩ part and only together they make functional enzyme. If plasmid is cleaved between them and insert is attached, then LacZ gene can´t produce β-galactosidase.
References:
http://en.wikipedia.org/wiki/Ortho-Nitrophenyl-β-galactoside
Time: 4 hours
Time in total: 46 hours 30 minutes
Ei kommentteja:
Lähetä kommentti