The Molecular Biology Notebook OnlineYou are here: Biotechnology > Gene Cloning > Plasmids
Cloning vectors and host cells
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.
- A plasmid is a small circular DNA which can reside in a bacterium, independently of it's genome. The plasmid, will not destroy the cell.
- The bacteriophage are viruses that infect bacteria and can multiply in a
bacterium, but, in some cases, will lyse the cell at the end of its cycle and release virus
particles into the medium. Note, some phages are non lytic (will not destroy the cell at the end of a cycle, it is the case of M13, and the lysogenic Lambda)
Both types have special properties that make them useful in molecular biology.
A typical plasmid
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.
Bacteriophages
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.
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Plasmids on Wikipedia
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