Biomedical Engineering Reference
In-Depth Information
of an uncut plasmid (which reveals the supercoiled plasmid species) or
the plasmid digested with a restriction enzyme, BamHI, that “linearizes”
the plasmid (cuts one time only) or a restriction enzyme, EcoRI, which
is the site into which the insert was cloned. Therefore, after cloning of
the insert into the plasmid, there are two EcoRI sites, one on either side
of the insert, and the insert “drops out” of the plasmid after digestion,
as shown on the gel. M identifies the marker lane, which is a ladder of
DNA fragments of know sizes. The marker can be used to calculate the
size of the insert.
Transformation
Once a recombinant plasmid is generated, you might be asking how
you get it back into the bacterium for its replication. Transformation is
the process by which bacterial or plasmid DNA is taken up by a bac-
terium. Luckily, some bacteria are naturally competent, that is they have
evolved ways to take up foreign DNA, such as plasmids. Haemophilus
influenzae and Bacillus subtilis are bacterial strains that have evolved
natural mechanisms to take up DNA. Bacteria can also be infected by
bacteriophages. These processes serve to increase their genetic com-
plexity and improve their survival (as mentioned earlier, plasmids often
carry antibiotic resistance genes), and to exchange pieces of their own
DNA with the introduced DNA (genetic recombination).
But what about bacteria that are not naturally competent? These
strains can be rendered competent experimentally. One bacterial strain
that is commonly used in the lab for recombinant DNA work is
Escherichia coli (E. coli). E. coli is a rod shaped gram negative bac-
terium with a circular chromosome that is approximately 3 million base
pairs in size. E. coli bacteria can be rendered competent by treatment
with salts (for example, calcium chloride) or by using a technique called
electroporation. Electroporation involves the use of a rapid electric pulse
to introduce DNA into a cell. This is thought to function by generating
pores in the membrane of the bacterium, which are then repaired. Elec-
troporation can be used for the introduction of DNA into eukaryotic cells
as well.
Bacteriophage
A bacteriophage is a virus that infects bacteria. Bacteriophages con-
tain sequences that direct the incorporation of their DNA into phage par-
ticles, which can then bind to and infect the bacterium. Bacteriophages
are useful as vectors since they generally can accommodate much
larger fragments of DNA than plasmids, and can still be successfully
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