Theory

Gene cloning is a fundamental technique in molecular biology that involves the replication of a specific gene to produce multiple identical copies. This process uses a vector, a DNA molecule that serves as a carrier, to introduce the gene of interest into a host cell. The gene is inserted into the vector to form recombinant DNA, which is then transferred into the host. The vector replicates with the inserted gene inside the host cell, generating numerous identical copies.

Vectors play a central role in gene cloning and expression. These specially designed DNA molecules transport foreign genes into suitable host cells. There are two primary types:

1. Cloning vectors, primarily used to replicate foreign DNA within a host.
2. Expression vectors replicate the DNA and direct the transcription and translation of the inserted gene, resulting in protein production.

Both types of vectors share basic features such as: An origin of replication (for autonomous replication in the host), Selectable markers (e.g., antibiotic resistance genes) for identifying transformed cells, A multiple cloning site (MCS) for gene insertion. However, expression vectors also include additional regulatory elements that enable and control gene expression. These include: A strong promoter (essential for initiating transcription), Enhancers and transcription terminators, A ribosome binding site, initiation codon, and stop codon. In E. coli, promoters in expression vectors are often inducible, allowing researchers to regulate gene expression using inducers (e.g., IPTG) or repressors.

The Green Fluorescent Protein (GFP), first isolated from the jellyfish Aequorea victoria, emits bright green fluorescence when exposed to blue or UV light. The GFP gene has become a widely used reporter gene due to its high stability, non-toxic nature, and ease of visualisation in live cells. Cloning and expressing the GFP gene in E. coli or other host organisms enables researchers to: Monitor gene expression in real time, Confirm successful gene insertion and transformation. Track protein localisation and interactions within cells.

Because GFP fluorescence is visible without additional substrates or cofactors, it is a convenient and powerful tool in molecular biology and biotechnology.