Cloning Vectors: Plasmids and Phages in Genetic Engineering

A cloning vector is a DNA molecule that can carry a foreign gene into a host cell and get it copied there. Plasmids and bacteriophages (phages) are the two classic vectors used in genetic engineering.

Vectors matter because a bare gene fragment cannot copy itself inside a cell and is easily lost. A good vector enters cells, replicates, carries selection markers, and holds the inserted gene safely for many generations.

Key Concepts

Plasmid is a small circular DNA in bacteria, separate from the chromosome. Origin of replication (ori) is where copying starts. Selectable marker, often an antibiotic resistance gene, identifies cells carrying the vector. Multiple cloning site has many enzyme cut sites for inserting genes. Bacteriophage is a virus that infects bacteria; lambda phage can carry larger DNA pieces than small plasmids. Copy number is how many vector copies exist per cell.

How It Works

1. Choose a vector suited to the size of the gene to be carried.
2. Cut vector and foreign DNA with matching restriction enzymes.
3. Join them with DNA ligase to form recombinant vector.
4. Introduce the vector into host bacteria.
5. Select hosts using the marker gene, for example growth on antibiotic medium.
6. Grow selected cells so the vector and inserted gene are copied together.

Worked Example

A plasmid has a copy number of 20 per cell. A culture reaches 500 million cells in 1 millilitre. Step 1: total plasmid copies per millilitre = 500,000,000 x 20 = 10,000,000,000 copies, which is 1 x 10 to the power 10. Step 2: if each plasmid carries one insulin gene copy, the culture holds the same number of gene copies per millilitre. Step 3: growing the culture to 10 litres (10,000 millilitres) gives about 10,000 x 1 x 10 to the power 10 = 1 x 10 to the power 14 gene copies, enough DNA for extraction and testing.

Common Misconceptions

• Plasmids are the bacterial chromosome. They are separate, smaller DNA circles; bacteria can live without many of them.
• Bigger vector is always better. Very large inserts can be unstable in small plasmids, which is why phages or other vectors are chosen for big DNA.
• All cells in a culture carry the insert. Some carry empty vector, so extra screening is used to confirm the gene is present.

Real-World Applications

Plasmids carry insulin, growth hormone and vaccine antigen genes in bacteria and yeast. Phage vectors help build genomic libraries that store an organism's DNA in pieces. Vectors are also used in gene therapy research and in producing genetically modified crops through plant-compatible vectors based on the Ti plasmid.

In brief, plasmids and phages act as delivery and copying vehicles for genes. Their ori, markers and cloning sites make cloning planned and selectable.