PROFESSOR: Welcome to
another help session
on recombinant DNA.
Today, we're going to
be discussing about
transformation and protein
expression.
As you can imagine, there are
often many times we will need
a large amount of protein.
But it can be difficult to get
it from the original source.
For example, you need insulin
to treat diabetes, but it's
not exactly practical to get a
lot of insulin from humans.
In order to get a lot of the
desired protein, often other
organisms will be used to
express this protein.
But it's a multi-step process.
For example, let's say we want
to express our human insulin
in bacteria.
Well, the human gene has both
introns and exons, as you
remember from lecture.
Exons are what are actually
cut together in order to
produce the final mature mRNA,
which is later used to express
the protein.
Bacteria, on the other hand,
don't have introns.
They just have exons.
So they are only capable of
reading a gene that just has
the exons and then producing
the protein from that.
In order to take a version of
the gene that's from the
eukaryotic cell and get it
to be expressed in the
prokaryotic, we first have to
make something called cDNA.
So let's begin.
We're going to take our
cell of interest.
And the first step in creating
the cDNA is we're going to
isolate the mRNA of interest--
this is the mRNA for insulin,
for example--
from the cell.
And once we have our mRNA, we
are going to add something
called reverse transcriptase.
This is a protein that's a DNA
polymerase that's going to use
the single-stranded RNA
as its template.
So it's going to take the RNA
and it's going to put together
this double-stranded DNA
that we're going
to refer to as cDNA.
So this is the DNA for the gene,
but unlike the original
gene, it only has the exons.
It doesn't have any
of the introns.
The next step, of course,
is to get the
cDNA into the bacteria.
We do this using something
called a vector.
The vector is just a means of
getting DNA into a cell.
One common type of vector
is a plasmid.
The plasmid is a circular piece
of DNA that the bacteria
can then take up and read.
The way we're going to get our
cDNA into this plasmid is
through the use of restriction
enzymes.
As you remember from our
previous help session,
restriction enzymes can cut up
DNA and create these overhang
of the nucleotides.
So we're going to cut up the
cDNA, and we're going to cut
up the plasmid, and they're
going to have overhangs that
are complementary.
This means that when we add
the cDNA to the plasmids,
they're going to hybridize,
and then
we can add DNA ligase.
We add DNA ligase, it will
create a phosphodiester bond
between the cDNA and the
plasmid vectors.
And finally we'll get the cDNA
inserted into our plasmid.
The next step, of course, is
getting the plasmid into the
bacteria in order for the
protein to be expressed.
There are multiple, different
ways to do this.
One common way is called
heat shock.
What happens is that the
bacteria is heated up and then
cooled rapidly, and this creates
lots of little holes
in the membrane, which
allow the plasma to
get into the cell.
Once you have the plasmid in
the bacteria, your job is
pretty much done.
Now the bacteria will naturally
express this
protein, so you can grow up
the bacteria in large
quantities and get a lot of
the protein of interest.
Referring back briefly to the
vector, there are several
parts that are important
for it to have.
We're going to need to have the
origin of replication, the
promoter, and the selection
marker.
The origin of replication
initiation, or ORI, is
necessary if we want the
bacteria to produce more
copies of this plasmid.
So once the plasmid gets into
the bacteria, if we don't have
an ORI, as the bacteria grows
and reproduces, none of the
daughter cells will
have this plasmid.
We'll have to continually
transform them.
However if it has the ORI,
as a bacteria grows and
reproduces, it will also
replicate this plasmid.
Another very important thing
to have is the promoter.
The promoter is a section of DNA
which signals for the RNA
polymerase to bind.
The RNA's polymerase will bind
to the promoter, and then will
proceed down the DNA on the
plasmid to read the actual
gene and transcribe it.
So the mRNA, which then
ultimately can
be made into protein.
Finally, you need a
selection marker.
Now as we talked about earlier,
when you heat shock
the bacteria, the plasmid
will get taken up.
However, not all the
bacteria might take
up some of the plasmid.
In order to get rid of the
unwanted bacteria, the
bacteria that doesn't have the
plasmid, we're going to use
selection marker.
A very common selection marker
for bacteria is antibiotic
resistance.
For example, if the plasmid
provides ampicillin
resistance, then this means that
any bacteria that takes
up the plasmid will be resistant
to ampicillin.
The bacteria that don't will
still be vulnerable to it.
So you could plate all of your
bacteria on a plate containing
ampicillin, and the ones that
have the plasmid will survive.
The ones that don't have the
plasmid will perish.
So let's go once more to the
original example and discuss
about what we're going to
need for our vector.
So again, we want to express
human insulin in
the bacterial system.
There are six possibilities
for what we
can need on our vector.
You can need the bacterial
ORI, the human ORI, the
bacterial promoter, the human
promoter, the bacterial
selection marker, the human
selection marker.
Pause for a minute.
Give you a chance to decide
what you think the vector
needs, and then we'll
go over it together.
OK.
Does it need a bacterial ORI?
Yes.
If we want to grow up a large
amount of insulin, we're going
to put the plasmid
in the bacteria.
The bacteria needs to create
more of this plasmid.
Does it need human ORI?
No, we're not actually assorting
the plasmid into a
human cell.
So the human cell is never
going to need to
create more of them.
Just the bacterial cell.
What about a bacterial
promoter?
Yes.
Even though it's a human
gene, the promoter has
to be for the bacteria.
Because it's going to be the
bacterial RNA polymerase that
will bind to the promoter,
and ultimately make the
mRNA from the cDNA.
What about a human promoter?
No, we don't need a human
promoter because the human RNA
polymerase won't be involved.
Again, this is only going to
be in the bacterial cells.
It's not going to be in the
human cell, so it doesn't need
a human promoter.
And finally for the selection
markers, once again, we only
need the bacterial selection
marker, not the human
selection marker.
We're not dealing with full
human cells at this point.
We're just dealing with a
plasmid, so we only need to
select for bacteria cells that
have the plasmid of interest.
This has been another help
section on recombinant DNA.
Thank you for your time.
