Plasmids: Definition, Types and Cloning Vectors

• A plasmid is a small, circular DNA molecule separate from a host cell's chromosome, often referred to as extra-chromosomal DNA.
• It replicates independently within a host cell, functioning as a replicon, which is a DNA fragment with replication components.
• Plasmids occur naturally in bacteria, yeasts, archaea, and some eukaryotic cells, forming symbiotic or parasitic relationships with their hosts.
• Plasmids carry genes responsible for specific traits in the host cell, with antibiotic resistance being the most common.
• They replicate prolifically, producing double-stranded fragments, making them useful as cloning vectors.
• A cloning vector is a DNA molecule that transports foreign DNA into a host cell, replicates, and generates numerous copies of itself and the foreign DNA.

History

• Independent DNA strands were discovered in bacterial cells in the late 1940s, but their nature was unclear initially.
• Joshua Lederberg coined the term "plasmid" in 1952, defining it as any extra-chromosomal hereditary element, during a study on Salmonella and virus P22.
• The transfer of bacterial genes by virus P22 to another host was identified, but the phenomenon remained unclear until the establishment of the double-helix DNA structure in 1952.
• Scientists have since discovered more plasmids, which are now heavily used in molecular and biotechnology research.

Plasmid Construction

• Construction involves using restriction enzymes to cut DNA at specific restriction sites.
• The resulting fragments are joined together using DNA ligase.
• Most plasmids are circular, their sizes range from less than 1.0 kilobase to over 250 kilobases.
• Small-sized plasmids utilize the host cell's enzymes for DNA replication.
• Larger plasmids can carry genes that code for replication enzymes.
• Some plasmids, known as episomes (especially in bacteria), insert themselves into the host's chromosome for replication.
• Some plasmids are linear, yet their replication is unique.

Origin of Replication

• Plasmids contain at least one DNA sequence where replication begins, known as the origin of replication (ORI).
• The ORI consists of 50-100 base pairs, enabling plasmid replication.
• Host-cell enzymes must bind to the ORI to start the replication process, common in circular plasmids.
• DNA replication continues around the circular plasmid once initiated, allowing any inserted DNA sequence to be replicated along with the plasmid DNA.
• The origin of replication ensures daughter cells have a copy of the plasmid after division.
• During plasmid replication, integration can be integrating or non-integrating.
• Non-integrating plasmids replicate independently without inserting into the host chromosome.
• Episomes insert themselves into the host chromosomes for replication

Plasmid Vectors

• Plasmid vectors are used in genetics and biotechnology to deliver traits into a host.
• They have a region for inserting a gene of interest, which is then introduced into a bacteria cell for replication.
• Vectors can contain drought-resistance and disease-resistance genes in crops, improving crop yields.
• Engineered plasmid vectors are smaller than naturally occurring plasmids, making them ideal carriers for gene transfer.
• Both plasmid vectors and naturally occurring plasmids are self-replicating, have an origin of replication, and carry unique genes like drug resistance genes.

Cloning

• Cloning involves inserting a gene into a host using a vector.
• The inserted gene is replicated in the host, displaying the desired characteristics.
• Plasmids are used as cloning vectors due to their small size, efficient origin of replication, and cloning limit of 100-10,000 base pairs.
• Some plasmids have multiple cloning sites for DNA insertion.
• Origin of replication, selectable markers, the gene of interest, and multiple cloning sites are used during cloning.

Protein Production

• Plasmid vectors are used to produce large quantities of proteins by inserting a gene responsible for production (e.g., the human insulin gene) into a plasmid vector, introducing it into a bacterium, the bacteria multiply, producing massive amounts of identical bacteria that produce the protein.

Gene Therapy

• Gene therapy involves inserting a healthy gene into human cells to cure or eliminate disease, treating genetic diseases by replacing or repressing defective genes.
• Genes are introduced into patients' cells using a plasmid cloning vector, administered through direct injection or intravenously.
• Gene therapy is used to treat cystic fibrosis, thalassemia, and hemophilia.

Disease Model

• Plasmids are used to generate disease models.
• Rat genetic models were initially created by engineering the embryonic stem cells of rats with plasmids.
• Human disease model creation is now possible by manipulating adeno-associated viruses using plasmids.

Recombinant Plasmid

• A recombinant plasmid results from inserting a gene of interest into a cloning vector, such as a plasmid.
• This plasmid acts as a carrier of the gene of interest to the host cell, leading to the expression of the gene in the host cells.

Selectable Marker

• Selectable markers, ensure that host cells contain the desired plasmid, such as antibiotic resistance genes.
• The vector carries a selectable marker to allow the selection of cells with the plasmid of interest.
• Cells are subjected to selective conditions, ensuring the survival of only those with the plasmid of interest.
• Genes conferring resistance to antibiotics are commonly used in cloning vectors to eliminate non-genetically modified bacteria.

Plasmid Example

• Virulence plasmids can turn a host, such as a bacterium, into a pathogen, making it a disease agent that can spread and replicate easily.
• Escherichia coli, naturally found in the human gut and other animals, can cause severe diarrhea and vomiting due to a virulence plasmid.
• Salmonella enterica also possesses a virulence plasmid.