Lac operon

The lac operon consists of three adjacent genes required for the transport and of lactose (milk sugar) in the Escherichia coli (E. coli) and some other bacteria. The term operon is used when genes (in this case lacZYA) are co-transcribed into a single messenger RNA. The lac operon is regulated by several factors, one of which is the availability of lactose as an energy source. Control of the lac genes was the first genetic regulatory mechanism to be elucidated, one reason for this is that it is one of the simplest, at least in outline, consisting of simple negative (lac repressor) and positive (CAP) regulatory elements. The lac operon has been considered the canonical example of prokaryotic gene regulation.

Lactose derivatives

A number of lactose derivatives or analogs have been described that are useful for work with the lac operon. These compounds are mainly substituted galactosides, where the glucose moiety of lactose is replaced by another chemical group. Isopropyl-?-D-thio-galactoside (IPTG) is frequently used as an inducer of the lac operon for physiological work.{{ref|Stryer}} IPTG binds to repressor and inactivates it, but is not a substrate for ?-galactosidase. The advantage of IPTG for in vivo studies is that it cannot be metabolized by E. coli, therefore the growth rate of cells (usually maintained with glycerol as the carbon and energy source), is not a variable in the experiment.

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Phenyl-?-D-galactose (phenyl-Gal) is a substrate for ?-galactosidase, but does not inactivate repressor and so is not an inducer. Since wild type cells produce very little ?-galactosidase, they cannot grow on phenyl-Gal as a carbon and energy source. Mutants lacking repressor are able to grow on phenyl-Gal. Thus, minimal medium containing phenyl-Gal is selective for repressor mutants and also for operator mutants.

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If a large number of cells (say, 108) of a wild type strain are plated on agar plates containing phenyl-Gal, the rare colonies which grow are mainly spontaneous mutants affecting the repressor. The relative distribution of repressor and operator mutants is affected by the target size, the number of different base pair changes which produce each type of mutation. Since the LacI gene encoding repressor is about 50 times larger than the operator, repressor mutants predominate in the selection.

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Other compounds serve as colorful indicators of ?-galactosidase activity. ONPG is cleaved to produce the intensely yellow compound, orthonitrophenol, and is commonly used as a substrate for assay of ?-galactosidase in vitro. X-gal (5-bromo-4-chloro-3-indolyl-?-D-galactoside) turns colonies which produce ?-galactosidase blue.

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Yet another analog, allolactose, is the "true" inducer of the lac operon. Lactose is galactose-(?1->4)-glucose, whereas allolactose is galactose-(?1->6)-glucose. Allolactose is produced by ?-galactosidase from lactose at a low level.

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A physiological experiment demonstrating this in E. coli cells is the observation that a null mutant of lacZ can still produce LacY permease when grown with IPTG but not when grown with lactose. The explanation is that processing of lactose to allolactose (catalyzed by ?-galactosidase) is needed to produce the inducer inside the cell.

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