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contain the foreign DNA fragment. The bacterial cells are then treated to open pores in
the cell membrane, which allows them to take up the recombinant plasmid. Once a bac-
terium has been transformed, it makes many copies of the recombinant plasmid, each
of which includes a copy of the foreign DNA. This is often called gene cloning since the
bacterium produces many identical copies (clones) of the original DNA fragment.
However, not all the bacterial cells will take up the recombinant plasmid. How can a
scientist or technician distinguish between bacteria with a plasmid and those without?
Plasmids used for transformation experiments often carry genes for antibiotic resistance,
which can then be used to select for transformed bacteria. By growing the bacteria in
medium that contains the antibiotic, any cells that do not contain a plasmid are killed off.
Individual bacteria cells are then grown into colonies so that the plasmid DNA can be iso-
lated from the cells and checked to make sure it contains the desired foreign DNA sequences.
For this transformation procedure to be successful, the plasmid DNA must have only
one recognition site for the restriction enzyme that is used, or else it would be cut into
a number of useless pieces. Naturally occurring plasmids do not always have a single
appropriate restriction enzyme site, so scientists have engineered plasmids especially
for transformation. Most of these engineered plasmids contain a multiple-cloning site,
which is a single region that contains unique recognition sites for an assortment of
restriction enzymes. The recognition sites are positioned very close together and are not
found anywhere else on the plasmid’s DNA sequence.
Vectors other than plasmids may also be used to transform bacteria, including viruses
and small inert particles that are literally fired into the cells.multiple-cloning site a region
in a vector that is engineered to
contain the recognition site of a
number of restriction enzymescircular plasmid DNA- Plasmid DNA is cut open at
EcoRI recognition site, producing
EcoRI sticky ends.
2. DNA ligase joins fragment
and plasmid.
Gene fragment to be inserted is cut out
from source using EcoRI; therefore, it has
EcoRI sticky ends.recombinant DNA- Plasmid is introduced into
bacterial cell.
bacterial
chromosomeplasmid with
foreign geneEcoRIFigure 9
A foreign gene is introduced into a
plasmid. The plasmid is now an
example of recombinant DNA,
which can be introduced into a
bacterial cell to produce numerous
copies of the gene.
DID YOU KNOW??
Plasmids: Beneficial Guests
Japanese scientists were the first to
discover plasmids that carry genes
for multiple drug resistance. The
bacterium Shigella, which causes
dysentery, developed resistance to
as many as four antibiotics,
including tetracycline, streptomycin,
chloramphenicol, and the
sulfonamides. The multidrug
resistance was due to a plasmid
within the bacterium that carried
genes for resistance and could be
passed naturally from bacterium to
bacterium.
How to Make cDNA
One way to make copies of a
particular gene is to use an
enzyme called reverse
transcriptase. This enzyme
synthesizes DNA from mRNA. The
resulting molecule is called copy
DNA or cDNA. The cDNA can
then be transferred into a vector
or a cell.http://www.science.nelson.com GO