Some Background

The lac operon is one of those great paradigms of gene expression - particularly of prokaryotes, which had its beginnings in the 1950s when Jacque Monod and Francois Jacob were studying E.coli and the expression of an enzyme, b-galactosidase, which allows E.coli to utilise lactose as a carbon source. The lac operon responsible for this was elucidated in 1961 (and was a finding that was rewarded by the Nobel Prize). Anyway, the important thing is that in the lac operon you have an inducible operator (a molecular switch that enables the gene to be ‘read’). When there is no lactose around (or lactose and glucose together as the glucose will be used before lactose), the product of the lac I gene, the lac repressor, is bound to the operator, which blocks binding of RNA polymerase and therefore preventing transcription of the lac genes (Z, Y and A).

...A stab at an answer :)

This property has been developed in biotechnology where the inclusion of specific lac genes into a cloning vector allows the identification of recombinant clones. A cloning vector may be a plasmid, which is inserted into a genetically engineered cloning host that does not posses the lac operon on its chromosome. This means that only those bacterial cells “transformed” with this plasmid will produce b-galactosidase. Now obviously, when cloning into a plasmid vector, you want to know whether your gene of interest as gone into your vector and ultimately into your cloning host. This is where methodologies such as pBluescript come in.

The lac operator is induced by the presence of lactose in the surrounding media, more specifically the lactose derivative allolactose. This binds to the operator bound lac repressor and causes an allosteric change that results in its release from the operator and allows transcription of the lac genes. In practice however, allolactose is not a good inducer as it is rapidly metabolised and repression is re- initiated.

Therefore, a synthetic inducer is used (often referred to as a gratuitous inducer) known as isopropyl-thiogalactoside (ITPG). What must be remembered however is that the ability to act as an inducer and the ability to act as a substrate are two different things. ITPG is a good inducer, but it does not tell us whether we have our plasmid of interest. What is needed is a chromogenic substrate such as X-gal. When this is hydrolysed by b-galactosidase, it produces a blue colour (5-bromo-4-chloro-3-indolyl-b-D-galactoside); or indeed there are substrates that turn yellow in the presence of b-galactosidase.

The selection of colonies containing the plasmid of interest can therefore be made by plating them onto media containing X-gal and ITPG. When you clone a gene into a vector, you design it to be cloned into a specific region of the plasmid, and this region is right in the middle of the lac Z gene (that encodes b-galactosidase). Therefore, any positive clones are not going to be able to hydrolyse X-gal and therefore they remain white. Those plasmids without inserts have a functional lac Z gene and are therefore blue.
Other chromogenic sugars now available permit black and white colour selection, where again, the white colonies contain the inserts of interest.

Further uses in Biotechnology

The application of such a controlled operator / repressor system has not been lost on biotechnology. The lac operator and repressor are also used in expression vectors. This is where you have cloned your gene into such a vector (though this time using another marker such as an antibiotic resistance cassette). These vectors, (the best known are called pET vectors) are transformed into specific hosts that contain on their genome the inducible lac operator, which is inserted upstream from a gene that encodes T7 RNA polymerase. There is also a lac I gene on the genome that produces the lac repressor, (which binds the operator).

The vector on the other hand contains a copy of the lac operator spliced together with a T7 promoter. This sequence is upstream from the target gene that you have cloned into your vector. Once you have selected those colonies containing plasmid vector + insert (via antibiotic selection etc.) you can think about inducing expression of your gene to produce whatever product it encodes, i.e. a protein.

By adding ITPG to this expression host the lac repressor is lost from the lac operators on both the host genome and the vector plasmid. This allows transcription of the T7 RNA Polymerase from the genome to take place. The RNA polymerase then binds to the free T7 promoter spliced onto the lac operator on the plasmid vector and transcription of the downstream target gene takes place - and because T7 is a very powerful promoter, virtually the whole cellular machinery becomes devoted to the expression of your protein.

..A conclusion (surely not!)

Certainly in this way we can produce large amounts of proteins / enzymes on which to conduct our studies. I cannot vouch for this at scaled industry level - nor can my colleagues, but I would assume that this approach is used to over-produce proteins in fermenters for sale at a commercial level.

Almost Done

There are numerous other methodologies by which the lacZ gene has been used as a reporter, especially with eukaryotic systems – with which I am less familiar.

There is also a-complementation, which is simply where you have a cloning host containing the lacZ gene (minus an ‘essential’ region), which produces an inactive dimeric form of b-galactosidase. Successful cloning can be detected if the cloned insert contains a peptide coding for the missing region and consequently in successful clones, the active tetrameric protein is formed and can be assayed.

There are other uses such as in mutagenesis and producing peptide libraries, but I think I have said enough. If you have further interest, I can recommend the reference [1] below. This is a good book on the history of the lac operon and can be read almost as a popular science text. I can highly recommend individual reading and study of the lac operon by bioscientists and interested non-scientists, as it is such an arresting and interesting paradigm in genetics and molecular biology.

J'ai fini!

Hope that helps?
Jim Caryl (very) MAD Scientist :-D

[1] Muller-Hill, B. (1996). The lac operon: a short history of a genetic paradigm. Walter de Gruyter & Co., Berlin.