Transcript Biotechnology part 2

Biotechnology Part 2
Insulin made by E.coli.
The last lecture we were talking about how to cut a gene out
of the host DNA and transfer it into a bacterial cell. We
ended with Gel Electrophoresis, to isolate the desired gene,
and PCR, to make many copies of the gene.
• 3. The plasmid vector is cleaved by same
restriction enzymes that were used to excise the
insulin gene.
– The reason that the same enzymes are used on both
is because they make identical sticky ends so gene
will fit and bind into plasmid.
• Imagine that you are cutting a strip of paper with patterned
scissors. If you were to cut one strip with one pattern and
another strip with a different pair of patterned scissors, the
two strips of paper would not match up with each other.
Restriction enzymes are essentially doing the same thing.
– The cut plasmid and the insulin gene are placed into
a test tube with each other and enzyme, ligase, is
added. The ligase is like a needle and thread. It is
responsible for binding the plasmid and gene sticky
ends together.
• 4. When the gene has been successfully
inserted into the plasmid vector, the vector is
then transformed into the target cell.
– Target cells are made competent, chemically or electrically, by
removing the cell wall.
– Competent cells (with the cell wall removed) are called
protoplasts.
– The vector is then added to a test tube full of competent cells.
– The competent cells can then take up or accept the extracellular
DNA.
– Next, the cells are grown in nutrient broth to promote healing and
growth for a short time before they are streaked on a plate for
colony separation.
• Notice that on the plasmid map in Fig. 10.8, there were a
couple of antibiotic resistance genes. These genes are
called reporter genes.
– Reporter genes help researchers know if the vector made it into
the cell quickly without having to isolate the plasmid.
• On the plasmid map one the genes is for
ampicillin resistance. So when the nutrient
plates are made for colony isolation of the
genetically engineered cells, some ampicillin is
added to the media. The ampicillin will kill any
cell that does not contain the plasmid for
ampicillin resistance.
– The ampicillin gene is the reporter gene because
when a cell survives and a colony is grown, we know
that the plasmid we inserted is present in the cell
because it is alive.
– Any other cells that don’t have the plasmid die
because of the ampicillin.
• Once a colony is identified with the
engineered plasmid, it is then grown in
large quantities.
• Then the protein product, in our case
insulin, is harvested.
Uses of genetically modified
organisms
• Microorganisms are used for making proteins
and enzymes and hormones because they grow
quickly and are easily manipulated.
– Not only that, but no one complains if thousands of
bacteria are killed for the sake of research or genetic
engineering.
• In addition to the proteins they make, sometimes
the genetically modified organisms themselves
are used.
– Ie. Bioremediation is the use of organisms that can
break down toxic chemicals. This is a great way to
clean up toxic spills.
– In addition, new microbes are engineered for pest
control for crops.
• However, here is something to think about.
– What happens when we introduce a new
microbe into the environment that never
existed before?
– How will its presence change the balance of
the ecosystem?
– Could we inadvertently create an organism
that might cause more harm to the
environment?
– On the other hand, so much good has and
can be done, should those endeavors stop
simply because we don’t know what the long
term effects (good or bad) will be?
Subunit vaccines are another example of the
importance of genetic engineering.
– The gene that causes disease for a particular
bacterial organism is placed in the vaccine.
– Advantage: no chance of becoming infected
with the disease because there is no
organism present in the vaccine.
– Disadvantage: time consuming, expensive,
and DNA is quickly degraded in the body.
• The homework for biotechnology is now
on the web. It will be due Friday, Oct. 13,
2006 by midnight.