Required for the transport and metabolism of lactose in Escherichia Coli (E. coli).The lactose operon or lac operon consists of three structural genes namely lacZ, lacY, and lacA that encode proteins involved in lactose metabolism as well as several regulatory genes.
E. coli grew in (differential medium) MacConkey agar. Image Credit: CA-SSIS / Shutterstock
What is lac operon?
An operon is defined as a functional unit of DNA that contains a group of genes under the control of same promoter. The lac operon in E. coli contains three structural genes, in addition to regulatory genes.
The structural genes include: lacZ – which encodes the enzyme, β-galactosidase; lacY – which encodes the enzyme, lactose permease; and lacA – which encodes the enzyme, lactose transacetylase.
A single promoter is responsible for the transcription of all these three genes, which produces a single mRNA to be translated into three different enzymes. Of these enzymes, β-galactosidase converts lactose into glucose and galactose; E. coli need β-galactosidase and lactose permease to properly utilize lactose for growth.
The regulatory genes of the lac operon include: lad (lac repressor) which contains its own promoter and terminator sequences; lacP (promoter) which can simply be described as the RNA polymerase-binding site; and lacO (operator) which is the repressor-binding site.
The lac repressor, which is produced from the lacl gene, regulates the transcription of the lac operon. The repressor binds to the lac operator to inhibit its transcription. On the other hand, the formation of the repressor-operator complex is prevented by the inducer, which is lactose in this case.
In other words, when lactose is present in the system, the repressor undergoes conformational changes, which result in a reduction of its binding affinity for the lac operator. As a consequence, the repressor dislodges from the operon and facilitates initiating gene transcription. Therefore, when E. coli grow on sources other than lactose, such as carbon-based sources, these genes are not transcribed, leading to a very low concentration of these enzymes in the cell. In contrast, the presence of lactose considerably increases the synthesis of these enzymes.
Besides the repressor-operator complex system, an additional control system does exist in E. coli to control the expression of lac operon genes. This additional system is called catabolite repression. This particular control system is based on the rate of glucose utilization by cells.
In other words, when both glucose and lactose are present in cells, transcription of lac operon genes is not initiated until all glucose molecules are used up. When cellular glucose concentrations are high, a glucose catabolite (glucose breakdown product) prevents the conversion of ATP into cAMP, which is a prerequisite for lac operon transcription initiation. Thus, glucose metabolism also regulates the expressions of lac operon genes by modulating the cellular concentration of cAMP.
Advantages and disadvantages of lac operon
The lac operon system of E. coli has been utilized widely for both experimental and industrial purposes. The lacZ gene, which encodes β-galactosidase, is used most extensively as a reporter gene in both prokaryotic and eukaryotic systems. It is considered as the most robust and reliable reporter system to track and monitor the products of lacZ genes.
The lacZ reporter assay is based on the ability of β-galactosidase to breakdown the colourless substrate X-gal into galactose and an insoluble product which is blue in colour. This assay can be used to visualize cells that express the reporter gene either constitutively or inducibly.
However, despite being a versatile reporter system, it has many limitations. For instance, this system is suitable for qualitative analysis of gene expression, however, quantification of the actual product needs further experimental procedures, such as cell harvesting and lysate preparation. This makes the system unsuitable for high-throughput screening. Moreover, the lacZ promoter is very large in size, thus, plasmids containing lacZ fusions are often very large and prone to deletion.
Besides the lacZ gene, the E. coli lac promoter has also been utilized extensively for research purposes. In addition to inducing the expression of homologous genes in the lac operon, it is also used to induce the expression of foreign genes that are cloned in a vector downstream to the lac promoter.
Isopropyl β-D-1-thiogalactopyranoside (IPTG), a molecular mimic of a lactose metabolite, is used to as an inducer to initiate gene transcription through the lac promoter.
However, the main drawback of this system is its inherent leakiness. This promoter maintains a basal level of gene expression even in the absence of an inducer. This makes this system unsuitable for many experimental conditions, especially if the gene of interest has toxic effects in cells.