We can genetically engineer things.
Now let us talk cloning. What is cloning?.
We were not talking about Dolly, the whole
sheep clone. We are talking about small pieces
of DNA. Cloning was initially the only way
we had to amplify or make more of a particular
piece of DNA. We may clone in order to create
a DNA library, a whole set of clones of all
of the DNA in an organism. And we may clone
in order to create more copies of a particular
piece of DNA, but it is a relatively slow
Towards the end of the lecture, I will introduce
polymerase chain reaction, which speeds the
whole thing up. But molecular cloning involves
isolation of the gene were interested in,
insertion of that gene into a bacterial genome
and then amplification by asking the bacteria
to amplify themselves as in reproduce and
make more copies of the genes we have inserted
into their plasmid or their bacterial chromosome.
We have a plasmid and this plasmid has two
special things in it, maybe three special things.
First of all, it needs an origin of replication.
Otherwise, we cannot make any copies of it.
If it has an origin of replication, then we
need to make sure that it has an inserted
antibiotic resistance gene. In addition to
that, we need a gene that expresses a particular
phenotype. Here we have the antibiotic gene
in red and this gene that expresses a particular
phenotype in yellow. In this case, we will
be looking at the lacZ gene, which codes for
the enzyme beta-galactosidase. Beta-galactosidase
breaks down the lactose sugars. If that gene
is functional, then betaglucosidase works,
if the gene is not functional, then it does
not work. Keep that in the back of your mind.
The ampicillin resistance gene is the red
piece and ampicillin resistance gene will
allow this plasmid or bacteria containing this plasmid
to grow when they are on an ampicillin-containing
medium. So now, we have the restriction endonuclease
that is going to cut specifically in the middle
of lacZ gene so that we can tell if the gene
of interest that we are inserting has been
inserted or not. So that lacZ gene has opened
up, we can then insert the foreign DNA.
It is going to want to anneal with the bacterial
plasmid and then we have a recombined plasmid.
Or we don't have a recombined plasmid perhaps
the DNA did not make it in there in which
case we have a functional betaglucosidase
gene and we have an interrupted one down on
the bottom with the new DNA of interest in
there. Now we need to get this DNA into a
bacteria so that the bacteria can reproduce
it. We are going to ask the bacteria to pick
up the plasmids from the environment. If they
do so, then we will take them to grow them
on a plate. Some of them keep in mind have
picked up the plasmid. Some of them have not
picked up the plasmid. Some of them have picked
up a plasmid that does not have the DNA of
interest in and some of them have picked up of
plasmid that does have the DNA of interest
in them. So we trick them with this plate of
media and the medium is going to contain ampicillin.
If the bacteria picked up the plasmid, then
it will have ampicillin resistance, the red gene
and they will grow on the plate. The other
thing that we supplement this plate with its X-gal.
X-gal is a sugar that when betaglucosidase is in
action, then it will turn blue. We have a
phenotypic thing we can observe. We will let
our bacteria that have been transformed grow
on this plate and if they have an active lacZ
gene, the blue color is formed and if they
do not have an active gene, then the bacterial
cultures grow white. From this result, we
can tell that not only has the bacteria taken
up the plasmid because otherwise they wouldn't
grow on the plate at all, the ampicillin will
kill them and also we can tell that it has
picked up gene of interest. So which culture
we really interested in on this plate? We are
interested in the ones that are depicted in
yellow that have the broken lacZ gene because
we know they have taken up the DNA of interest, whatever
that might be. We will look at some examples
shortly. So now,
let us examine a few different host-vector