Cambridge International A Level Biology Revision Guide

Cambridge International A Level Biology
466
is used that gives blunt ends, then sticky ends need to be
attached to both the gene and the plasmid DNA.
The opened plasmids and the lengths of DNA are
mixed together. Some of the plasmid sticky ends pair
up with the sticky ends on the new gene. The enzyme
DNA ligase is used to link together the sugar–phosphate
backbones of the DNA molecule and the plasmid,
producing a closed circle of double-stranded DNA
containing the new gene. This is now recombinant DNA.
Bacterial plasmids can be modified to produce good
vectors. Plasmids can also be made artificially. For
example, the pUC group of plasmids have:
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a low molecular mass, so they are readily taken up by
bacteria
an origin of replication so they can be copied
several single target sites for different restriction
enzymes in a short length of DNA called a polylinker
one or more marker genes, allowing identification of
cells that have taken up the plasmid.
Plasmids are not the only type of vector that can be
used. Viruses can also be used as vectors. A third group
of vectors are liposomes, which are tiny spheres of lipid
containing the DNA. There is more about these other
vectors later in this chapter.
Getting the plasmids into bacteria
The next step in the process is to get bacteria to take up
the plasmids. The bacteria are treated by putting them
into a solution with a high concentration of calcium ions,
then cooled and given a heat shock to increase the chances
of plasmids passing through the cell surface membrane.
A small proportion of the bacteria, perhaps 1%, take up
plasmids with the gene, and are said to be transformed.
The rest either take up plasmids that have closed without
incorporating a gene or do not take up any plasmids at all.
Identifying bacteria with recombinant DNA
It is important to identify which bacteria have been
successfully transformed so that they can be used to make
the gene product. This used to be done by spreading the
bacteria on agar plates each containing an antibiotic. So
if, for example, the insulin gene had been inserted into the
QUESTION
19.2 Summarise the advantages of using plasmids as
vectors in genetic engineering.
plasmid at a point in the gene for tetracycline resistance in
pBR322, then any bacteria which had taken up plasmids
with the recombinant DNA would not be able to grow on
agar containing tetracycline. However, this technique has
fallen out of favour, and has largely been replaced by simpler
methods of identifying transformed bacteria (page 468).
DNA polymerase in bacteria copies the plasmids; the
bacteria then divide by binary fission so that each daughter
cell has several copies of the plasmid. The bacteria
transcribe the new gene and may translate it to give the
required gene product, such as insulin.
Insulin production
One form of diabetes mellitus is caused by the inability
of the pancreas to produce insulin (Chapter 14). Before
insulin from GM bacteria became available, people with
this form of diabetes were treated with insulin extracted
from the pancreases of pigs or cattle. In the 1970s,
biotechnology companies began to work on the idea of
inserting the gene for human insulin into a bacterium
and then using this bacterium to make insulin. They tried
several different approaches, finally succeeding in the early
1980s. This form of human insulin became available in
1983. The procedure involved in the production of insulin
is shown in Figure 19.5.
There were problems in locating and isolating the gene
coding for human insulin from all of the rest of the DNA
in a human cell. Instead of cutting out the gene from the
DNA in the relevant chromosome, researchers extracted
mRNA for insulin from pancreatic β cells, which are the
only cells to express the insulin gene. These cells contain
large quantities of mRNA for insulin as they are its only
source in the body. The mRNA was then incubated with
the enzyme reverse transcriptase which comes from
the group of viruses called retroviruses (Chapter 10,
page 206). As its name suggests, this enzyme reverses
transcription, using mRNA as a template to make singlestranded
DNA. These single-stranded DNA molecules
were then converted to double-stranded DNA molecules
using DNA polymerase to assemble nucleotides to make
the complementary strand. The genetic engineers now
had insulin genes that they could insert into plasmids to
transform the bacterium Escherichia coli.
The main advantage of this form of insulin is that there
is now a reliable supply available to meet the increasing
demand. Supplies are not dependent on factors such as
availability through the meat trade.