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PROFESSOR: Welcome to
another help session

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on recombinant DNA.

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Today, we're going to
be discussing about

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transformation and protein
expression.

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As you can imagine, there are
often many times we will need

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a large amount of protein.

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But it can be difficult to get
it from the original source.

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For example, you need insulin
to treat diabetes, but it's

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not exactly practical to get a
lot of insulin from humans.

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In order to get a lot of the
desired protein, often other

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organisms will be used to
express this protein.

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But it's a multi-step process.

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For example, let's say we want
to express our human insulin

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in bacteria.

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Well, the human gene has both
introns and exons, as you

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remember from lecture.

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Exons are what are actually
cut together in order to

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produce the final mature mRNA,
which is later used to express

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the protein.

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Bacteria, on the other hand,
don't have introns.

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They just have exons.

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So they are only capable of
reading a gene that just has

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the exons and then producing
the protein from that.

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In order to take a version of
the gene that's from the

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eukaryotic cell and get it
to be expressed in the

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prokaryotic, we first have to
make something called cDNA.

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So let's begin.

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We're going to take our
cell of interest.

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And the first step in creating
the cDNA is we're going to

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isolate the mRNA of interest--

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this is the mRNA for insulin,
for example--

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from the cell.

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And once we have our mRNA, we
are going to add something

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called reverse transcriptase.

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This is a protein that's a DNA
polymerase that's going to use

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the single-stranded RNA
as its template.

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So it's going to take the RNA
and it's going to put together

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this double-stranded DNA
that we're going

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to refer to as cDNA.

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So this is the DNA for the gene,
but unlike the original

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gene, it only has the exons.

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It doesn't have any
of the introns.

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The next step, of course,
is to get the

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cDNA into the bacteria.

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We do this using something
called a vector.

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The vector is just a means of
getting DNA into a cell.

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One common type of vector
is a plasmid.

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The plasmid is a circular piece
of DNA that the bacteria

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can then take up and read.

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The way we're going to get our
cDNA into this plasmid is

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through the use of restriction
enzymes.

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As you remember from our
previous help session,

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restriction enzymes can cut up
DNA and create these overhang

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of the nucleotides.

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So we're going to cut up the
cDNA, and we're going to cut

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up the plasmid, and they're
going to have overhangs that

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are complementary.

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This means that when we add
the cDNA to the plasmids,

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they're going to hybridize,
and then

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we can add DNA ligase.

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We add DNA ligase, it will
create a phosphodiester bond

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between the cDNA and the
plasmid vectors.

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And finally we'll get the cDNA
inserted into our plasmid.

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The next step, of course, is
getting the plasmid into the

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bacteria in order for the
protein to be expressed.

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There are multiple, different
ways to do this.

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One common way is called
heat shock.

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What happens is that the
bacteria is heated up and then

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cooled rapidly, and this creates
lots of little holes

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in the membrane, which
allow the plasma to

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get into the cell.

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Once you have the plasmid in
the bacteria, your job is

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pretty much done.

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Now the bacteria will naturally
express this

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protein, so you can grow up
the bacteria in large

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quantities and get a lot of
the protein of interest.

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Referring back briefly to the
vector, there are several

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parts that are important
for it to have.

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We're going to need to have the
origin of replication, the

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promoter, and the selection
marker.

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The origin of replication
initiation, or ORI, is

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necessary if we want the
bacteria to produce more

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copies of this plasmid.

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So once the plasmid gets into
the bacteria, if we don't have

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an ORI, as the bacteria grows
and reproduces, none of the

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daughter cells will
have this plasmid.

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We'll have to continually
transform them.

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However if it has the ORI,
as a bacteria grows and

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reproduces, it will also
replicate this plasmid.

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Another very important thing
to have is the promoter.

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The promoter is a section of DNA
which signals for the RNA

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polymerase to bind.

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The RNA's polymerase will bind
to the promoter, and then will

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proceed down the DNA on the
plasmid to read the actual

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gene and transcribe it.

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So the mRNA, which then
ultimately can

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be made into protein.

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Finally, you need a
selection marker.

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Now as we talked about earlier,
when you heat shock

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the bacteria, the plasmid
will get taken up.

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However, not all the
bacteria might take

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up some of the plasmid.

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In order to get rid of the
unwanted bacteria, the

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bacteria that doesn't have the
plasmid, we're going to use

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selection marker.

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A very common selection marker
for bacteria is antibiotic

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resistance.

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For example, if the plasmid
provides ampicillin

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resistance, then this means that
any bacteria that takes

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up the plasmid will be resistant
to ampicillin.

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The bacteria that don't will
still be vulnerable to it.

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So you could plate all of your
bacteria on a plate containing

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ampicillin, and the ones that
have the plasmid will survive.

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The ones that don't have the
plasmid will perish.

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So let's go once more to the
original example and discuss

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about what we're going to
need for our vector.

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So again, we want to express
human insulin in

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the bacterial system.

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There are six possibilities
for what we

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can need on our vector.

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You can need the bacterial
ORI, the human ORI, the

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bacterial promoter, the human
promoter, the bacterial

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selection marker, the human
selection marker.

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Pause for a minute.

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Give you a chance to decide
what you think the vector

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needs, and then we'll
go over it together.

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OK.

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Does it need a bacterial ORI?

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Yes.

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If we want to grow up a large
amount of insulin, we're going

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to put the plasmid
in the bacteria.

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The bacteria needs to create
more of this plasmid.

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Does it need human ORI?

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No, we're not actually assorting
the plasmid into a

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human cell.

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So the human cell is never
going to need to

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create more of them.

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Just the bacterial cell.

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What about a bacterial
promoter?

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Yes.

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Even though it's a human
gene, the promoter has

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to be for the bacteria.

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Because it's going to be the
bacterial RNA polymerase that

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will bind to the promoter,
and ultimately make the

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mRNA from the cDNA.

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What about a human promoter?

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No, we don't need a human
promoter because the human RNA

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polymerase won't be involved.

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Again, this is only going to
be in the bacterial cells.

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It's not going to be in the
human cell, so it doesn't need

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a human promoter.

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And finally for the selection
markers, once again, we only

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need the bacterial selection
marker, not the human

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selection marker.

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We're not dealing with full
human cells at this point.

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We're just dealing with a
plasmid, so we only need to

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select for bacteria cells that
have the plasmid of interest.

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This has been another help
section on recombinant DNA.

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Thank you for your time.