For prokaryotes, genes are polycistronic – expression of multiple gene is mediated by one promoter.
In bacteria such as E. coli, it appears that the products of carbohydrate metabolism themselves activate the switch between glucose and lactose use.
When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to the lac repressor and makes it change shape so it can no longer bind DNA. Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or operon.
Thus, RNA Polymerase will bind with promoter region of gene and starts transcription.
On the other hand when lactose is absent, lac repressor binds with operator preventing RNA Polymerase to perform any further transcription.
1. If Both Glucose and Lactose are Present
E. coli prefers to use glucose over lactose for energy. When glucose is present, it is metabolized first. The breakdown of glucose produces catabolites. These catabolites prevent the production of cyclic AMP (cAMP), a crucial signal molecule. The absence of cAMP means that the catabolite activator protein (CAP) cannot form the CAP-cAMP complex. Without the CAP-cAMP complex, CAP cannot bind to the DNA sequence near the lac operon. This lack of binding prevents the activation of the lac operon, even if lactose is present. Enzymes required for lactose metabolism are not produced. E. coli continues to use glucose and does not metabolize lactose.
2. If Glucose is Absent and Lactose is Present
When glucose is not available, E. coli needs to switch to using lactose. The absence of glucose catabolites leads to an increase in cAMP levels. Increased cAMP binds to catabolite activator protein (CAP), forming the CAP-cAMP complex. This complex then binds to a specific DNA sequence near the lac operon operator and promoter. The binding of the CAP-cAMP complex enhances the binding of RNA polymerase to the lac operon. This leads to the activation of gene expression from the lac operon. If lactose is present, it binds to the lac repressor (lac i), inactivating it and allowing transcription of the lac operon. Enzymes necessary for lactose metabolism are produced. E. coli can now metabolize lactose in the absence of glucose.
Induction of IPTG is used for overexpression of Protein
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a molecular mimic of allolactose, a lactose metabolite, and is commonly used to induce protein expression in bacterial systems, particularly in E. coli. IPTG is used to induce the expression of genes that are under the control of the lac operon.
The lac operon is a set of genes regulated by lactose, which includes a promoter, an operator, and structural genes. IPTG acts by binding to the lac repressor, preventing it from inhibiting the lac operon, thereby allowing gene expression.
To achieve targeted protein production, we will create a mutant strain by replacing the Lac Z, Y, and A genes with our gene of interest using an expression vector. In the presence of IPTG, the lac operon will be functional, and our gene of interest will be expressed, producing the desired enzyme. Importantly, IPTG, as a gratuitous inducer, will not be metabolized by the cells and will remain active throughout the process.
Why IPTG instead of Lactose?
IPTG is a gratuitous inducer (a molecule that induces the expression of specific genes without being metabolized by the cell)
Unlike lactose, IPTG doesn’t get utilized by enzyme that are produced by Lac Operon.
Applications:
- For analyzing and studying
- Assisting in cure
- Determining enzymatic mechanism
- Producing life saving products
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