BAC: Super-Sized Inserts

Bacterial Artificial Chromosomes (BAC) have been developed to hold much larger pieces of DNA than a plasmid can. BAC vectors were originally created from part of an unusual plasmid present in some bacteria called the F’ plasmid.

The F’ plasmid allows bacteria to have “sex” (well, sort of: F’ helps bacteria give its genome to another bacteria but this only happens rarely when bacteria are under a lot of stress). F’ had been studied extensively and it was found that it could hold up to a million basepairs of DNA from another bacteria. Also, F’ has origins of replication and bacteria have a way to control how F’ is copied.

Genetic Carrying Handles: Cloning Vectors

In order to clone a gene, its DNA sequence must be attached to some kind of carrier, also made of DNA, that can take it into the cell. Biologists call these carriers vectors. A vector acts like a handle for the DNA, and it also contains other tools such as an origin of replication and a selective marker.

The origin of replication is a sequence of DNA that the host cell recognizes that allows it to make more copies of the clone DNA sequence. This origin sequence is where the cell begins copying the vector and the attached clone DNA. The selective marker is a specific DNA sequence that is used by biologists to tell if the clone has entered the cell, and they are usually genes that confer antibiotic resistance to the cell.

The most common media used for this process is actually very similar to chicken soup, but the carbohydrate agarose is added to convert the media into a semi-solid substance, since bacterial colonies are much easier to detect on a semi-solid surface. Agarose is much like gelatin, but it comes from seaweed and unlike gelatin, most bacteria cannot digest it. Antibiotics are often added to the media, to kill any cells that do not possess the antibiotic resistant selective marker gene, which is in the clone DNA. This way, biologists can ensure that all the remaining cells have in fact taken up the clone DNA and its vector.

These cells are called transfected cells. Antibiotics are not the only way to identify transfected cells. Biologists sometimes use selective markers that turn cells a different colour or even to make them glow. Common proteins that do this include luciferase, which makes fireflies glow or green fluorescent protein, which comes from certain species of jellyfish. Green fluorescent protein also comes in other colours!

Bacterial artificial chromosome

A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150 kbp, with a range from 100 to 300 kbp. A similar cloning vector, called a PAC has also been produced from the bacterial P1-plasmid.

BACs are often used to sequence the genetic code of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism.