The vector is the main participant in the cloning game. It is a fragment of DNA, usually circular, carrying the necessary genes that tell the host to multiply it, and some other genes which are used as controls in the experiments.
Two types of DNA can replicate in Bacteria: Plasmids and Bacteriophages.
Both types have special properties that make them useful in molecular biology.
A plasmid needs a replication origin: it is necessary for the replication of the plasmid. The DNA replication machinery of the host cell needs this signal to initiate replication. If it is not there, or can't be recognised, the plasmid will not be copied.
It is necessary to determine if, after the transformation of the host
cell, the bacterium has a plasmid or not. The simplest way to do
this is to design a plasmid that will give a bacterium resistance to an antibiotic like Ampicillin. If the bacterium possesses such a plasmid, it will survive
in a medium containing Ampicillin, an antibiotic commonly used in molecular
biology. If the plasmid is not present, the bacterium will not grow. It
is not dead, but dormant.
After the transformation, the solution containing bacterium and plasmid, is put onto a plate which contains Ampicillin. If a plasmid has penetrated a host cell, it gives it resistance to ampicillin and the cell will grow.
Usually, one infects bacteria with plasmids in order to clone pieces
of DNA. It is important to know if such a clone
of bacteria has a plasmid with an insert or not as it can happen that the recombination of DNA has not worked with 100% efficienty. It is important to be able to discern which clones contain a plasmid with an insert and which contain a plasmid with no insert.
DNA can be inserted into the plasmid in the middle of a gene coding for an enzyme that hydrolyses galactose Galactose, or similar compounds, are not usually used by a bacterium, but are when an inducer is present. When the inducer is present, and the enzyme works, it will hydrolyse galactose, or any galactoside. Xgal is a galactoside that is colourless, but once hydrolysed is blue. If the enzyme is present and fully working, providing that an inducer and some Xgal are added, then the colony will be stained blue. If the enzyme is not working, then the colony will be white. If a piece of DNA, however small is introduced into the gene that codes for the specific enzyme, it won't be expressed, therefore there will not be any enzyme to degrade the dye and the colony will remain white. This is a good test for determining the presence of an insert in a plasmid. Note that there are many other ways to detect the presence of a recombinant plasmid in a bacterium, and this is just one example.)
A run through an experiment is given as an example.
Plasmids are the most likely cloning vectors in the bacterium E. coli but they are not the only ones. Vectors derived from the bacteriophage Lambda are often used to clone bigger fragments. The cloning methods with a phage Lambda are different because a phage produces plaques, which are areas where the bacteria have been lysed, or destroyed, and not colonies.
A commonly used bacteriophage is Lambda. Lambda is a temperate phage. This means it has a choice between a lytic or a lysogenic (cryptic) cycle. In the lytic cycle, the infection of the bacteria is immediatly followed by taking over of the bacterium's machinery to synthetise capsule's proteins and replicate the virus genome, and lyse of the bacterium. In the lysogenic cycle, the phage DNA is incorporated in the bacterium's genome and hidden (cryptic) there until an event (heat, or UV) triggers a lytic cycle. The bacterium might have had the time to divide in the mean time, multiplying the number of copies of the phage.