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We're going to talk today about
recombinant DNA.

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This is a new section of the class.
We'll spend three lectures talking

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about recombinant DNA,
just sort of a practical how-to

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guide with respect to manipulating
DNA now that you've learned a bit

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about it and how we use it in
research and in other aspects of

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medicine and agriculture and so on.
Now, recombinant DNA has been around

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for a long time.
And, frankly, it's got a bit of a

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bad rap.  When people in the general
public hear the term recombinant DNA

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they sort of conjure up these ideas
of mad scientists going around,

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you know, cloning bizarre organisms
or creating dangerous

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forms of life.
Although that's possible,

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it's not what we typically do with
this technology.

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In fact, it has some rather benign
and other quite important

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applications.  So,
Claudette, I'm not going to get this

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board, am I?  OK.

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Basically what we're going to be
talking about is genetic engineering.

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That ought to make the budding
engineers in the crowd happy.

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We're going to manipulate the DNA
of organisms and actually transfer

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DNA from one organism to another for
various purposes.

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There are three general tenants
that you need to pay attention to

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here.  The basic goal is to isolate,
from a complex genome, a piece of

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DNA of interest.
And amplify that piece of DNA,

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which could be a specific gene that
you're interested in,

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or it might be some other sequence
within that DNA.

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It doesn't have to necessarily be a
coding gene that you're interested

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in.  The purposes of this are to,
for example, produce therapeutic

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proteins or compounds.
In the context of disease,

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for example, where individuals have
a genetic disease in which they

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cannot make a particular enzyme,
you might be able to treat that

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disease by producing the relevant
enzyme via this technology in

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another organism and then supply it
to that individual.

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The organism that you put it in
could be a bacterium that produces

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it in large quantities.
And you purify the enzyme and then

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perhaps inject it into the
individual.  It doesn't have to be

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bacteria.  You can actually use
recombinant DNA technology,

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genetic engineering to produce a
human protein in,

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for example, some farm animal.
And there are actually companies

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that will introduce human genes into
sheep or cows such that you can

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purify the recombinant protein from
the milk of that organism.

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And perhaps it might be more folded
more properly or modified more

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properly than it would be if in
bacteria.  And it might be more

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easily purified from the milk than
it would be from the bacterial cells.

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We can also engineer organisms.

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So this is to make different life
forms that have different purposes,

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different values to us.  And I've
given you the example in past

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lectures about genetically modified
foods.  And your book talks

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about this as well.
You can make crops that are

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resistant to pesticides.
You can make crops that are

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resistant to herbicides.
You can make crops that stay

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fresher longer,
taste better, have better shape for

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packing, all sorts of things by
manipulating the genome of those

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organisms such that they now have
new and useful properties.

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We'll also teach you about how to
make transgenic animals.

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I've given you one example of this
already where you might want to make

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a therapeutic protein in the milk of
a transgenic cow,

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but there are lots of purposes for
making transgenic animals.

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One important one, especially for
me, is to make disease models.

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So once we understand a human
genetic disease we can use that

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information to create animal species,
most typically mice that carry those

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very same mutations.
And then study the disease process

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in the context of a laboratory mouse
in ways that are difficult or

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impossible to do in humans.
And we'll teach you how to do that.

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It all flows from the same general
principles of manipulating DNA and

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transferring it from one organism to
another.  The value of doing this is

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several-fold.

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Understanding our or other
DNA sequences.

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So without this technology,

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we actually couldn't have done the
genome project.

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We need to purify up a lot of our
DNA in order to sequence it to

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determine its sequence.
And likewise of other organisms.

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So this was instrumental in
bringing us to the point we are

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today of understanding our genetic
information to the nucleotide level.

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As I mentioned, this technology
allows us to understand

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disease-causing mutations.

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Again, we've taught you about that
mutations can occur,

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disabled enzymes or other proteins
cause disease,

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but that doesn't tell you why.
And using this technology to sort

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of dissect what's happening to that
individual gene by manipulating it

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in different ways,
putting it in different contexts we

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can begin to understand what the
specific consequences of that

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mutation are, which ultimately
allows us to deal with the disease

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more effectively.
And another value of just

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manipulating DNA,
which we'll come to in a future

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lecture, is what you hear about in
the press as DNA fingerprinting.

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It's a very practical use of this
technology in which you want to

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distinguish two individuals based on
the specific changes in their DNA.

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And we can do that because we can
amplify up DNA sequences from very,

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very small quantities and figure out
the specific nucleotide sequences of

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those DNA fragments and be able to
tell then two people apart.

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And this is useful in paternity
cases, it's useful in criminal cases,

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it's useful for archeological
purposes and otherwise.

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So there are really a great number
of values to this set of

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technologies.  So where do they come
from?  Well, we've been able to do

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this sort of thing since the late
1960s, early 1970s.

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And this is referred to,
in our field, as the Recombinant DNA

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Revolution

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And there were three things that
happened in this time period that

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made this technology possible.
Firstly was the ability to separate

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genomic DNA, specifically
into fragments.

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So genomic DNA,
as you know, is huge.

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Our chromosomes are long,
long linear pieces of DNA.  Even

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bacterial chromosomes are a few
million nucleotides in length.

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That's much too much to deal with
for many of these applications.

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And so it was necessary to
basically chop the genomic DNA up

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into manageable sized pieces and
then be able to deal with those

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pieces individually.
The second thing that

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was necessary --

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-- was the ability to transfer the
isolated pieces --

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-- into another organism.

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And the organism of choice then and
largely now is bacteria.

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So it was observations made in the
1960s that bacteria would actually

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take up from their environment
pieces of DNA and sometimes

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incorporate them within
their cells.

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That observation led to the notion
that you could do that on purpose,

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not just at random, and that would
be then using the bacteria basically

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as a cargo ship for the introduced
DNA.  And, finally,

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the ability to amplify the fragments
of interest to large quantities.

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So if I take one piece of DNA from

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this guy, it's a single molecule,
that's not enough.  I cannot use

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that for much.
I cannot use it to sequence his DNA,

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for example.  I cannot use it to
study specific properties of that

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gene.  Instead I need to amplify it
up to very large quantities.

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And, therefore,
it was necessary to figure out ways

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basically to turn the bacteria into
little DNA factories that would

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churn out large quantities of DNA of
interest.  And,

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again, we'll review how that was
possible.  And all of these things

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came together more or less the same
time of this timeframe,

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and then the field took off.
Now we could manipulate DNA very

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precisely and begin to understand
DNA at a very specific level.

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So I'm going to give you some
examples of what we do.

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I'm actually going to give you
perhaps the first and only practical

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demonstration of this.
We're going to do an experiment

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before you, a dangerous thing to do
in front of a live audience.

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We're actually going to transfer a
gene from one organism to another.

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We're going to start it today and
we'll finish it in subsequent

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lectures.  The gene of interest is a
toxic gene.  It's a toxic gene

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that's present in an organism known
as S. pyogenes.

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You might know of this organism,
might have heard of this organism

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because it's the flesh eating
bacterium.  Anybody heard of the

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flesh eating bacterium?
This is true.  If you get infected

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with S. pyogenes and you're slightly
immunocompromised,

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but even if you have a bad strain of
it even if you're very healthy,

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the bacterium will go about eating
your flesh.  It actually can be

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lethal it's so devastating.
The organism, S.

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pyogenes is a bacterium that has a
genome, of course.

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That genome has about a thousand
genes.  And the sequence of this

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genome is known.
So by standard DNA sequencing

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technologies, we'll actually review
those for you in a couple of

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lectures, specific DNA sequences of
this organism is known.

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OK?  So we know exactly which genes
there are there.

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And, just for the sake of discussion,
let's consider a couple of genes.

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Gene A, gene B and gene T.  And
it's this gene that we're interested

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in.  It's the gene that encodes a
very critical toxin that the

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bacterium makes that kills your
cells.  And so when you're badly

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infected by this bacterium,
it's dumping out this toxin and it's

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killing the cells in the surrounding.
And that's why it's

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flesh eating.  OK?
Now, you also heard in the previous

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couple of lectures ago that these
genomes have other important

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properties.  And just to remind you
they have origins of replication,

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ORIs.  All genomes, for the purposes
of replication,

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all pieces of DNA that are going to
be replicated need at least one

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origin of replication.
So this genome has one.

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And this will come up in another
context shortly,

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but what we're really interested in
is this gene T here,

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the toxin gene.  We want to clone it.
We want to move it from this

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organism to another organism.
And specifically the other organism

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of choice, and this is often the
case, is E. coli.

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E. coli is a common gut bacterium
present in all of you in very,

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very large quantities.  It has been
modified for laboratory purposes

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largely weakened so that it cannot
easily make its way back into you

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and cause possible harm.
It's used in laboratories in

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another context all the time.
I'm depicting here an E.

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coli cell.  This is always confusing
because I depicted here with a very

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similar circle the genome of S.
pyogenes.  This is a bacterial cell.

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This is the membrane of the
bacterium.  And,

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of course, E. coli has its own
genome which has roughly the same

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size and roughly the same numbers of
genes.  What we're going to do is

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transfer one more gene from this
organism into that organism.

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Really, the purpose of today's
lecture is how do you do that?

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But before we consider how you do
it, let's ask the question why you

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would want to do it.
Anybody have any ideas?

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Why would you want to transfer this
potentially deadly toxin producing

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gene to another organism?  Yeah?
It could be that in a different

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context it's actually beneficial in
one organism.  You may be able to

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make a better E.
coli for some purpose if you

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transferred this gene.
My favorite idea, actually,

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is global terror.

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We can laugh about this but,
you know, there are terrorists

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around who are thinking these very
thoughts.  Sad but true,

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people are making modified organisms
with dangerous genes to make them

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more ìpathogenicî for deliberate
release into populations.

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So I'm not teaching you how to do
this for malicious purposes but just

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to know that there are people who do
that sort of thing.

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Related to this point over here,
we want to understand these genes

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for the purposes of biological
research.  We want to understand as

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much as we can about disease-related
properties of genes and so on.

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And sometimes you cannot do the
experiments you want to do in the

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organism in which the
disease occurs.

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And it's easier to do it,
in a sense, in isolation in another

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organism.  And then another example
might be to produce a vaccine.

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If I could produce this protein
that's so toxic in large quantities,

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I might be able to make a version of
it which is not toxic,

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very similar but not toxic.
And then I could inject it into

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people, they would raise an antibody
response against it such that if

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they were ever challenged with the
real dangerous guy they

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would be immune.
So that would be a very useful thing

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to do.  And, again,
if we were worried about the

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terrorists we might do exactly that.
So this is the goal of our

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experiment.  And we're going to
start the experiment today.

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So what I've done is acquired from
one of my colleagues in the Biology

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Department two tubes in which we
have placed these bacteria.

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S. pyogenes is in this one.
E. coli is in this one.  There are

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bacterial cells in here that grow in
suspension.  They grow inside

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the liquid medium.
If we want to isolate the DNA we

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first have to isolate the bacteria.
So we spin these tubes in a

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centrifuge.  The bacteria then
pellet to the bottom of the tube.

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So the bacteria are now collected
at the bottom of these tubes.

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We then decant off the liquid.
I meant to bring a beaker, but I

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don't think they'll mind if I use
this one.  So it's always a little

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bit dangerous to do this,
and we'll have to take care of this

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later because it is a bit dangerous.
It's always a little bit dangerous

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to do this because you don't want to
bump yourself like I just did.

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00:17:05,000 --> 00:17:13,000
It actually is starting to hurt a
little bit.  So we'll get rid of the

224
00:17:13,000 --> 00:17:21,000
liquid here.  And then we'll add
various chemical solutions to

225
00:17:21,000 --> 00:17:29,000
isolate the DNA from this bacterium.
Wow.  It's really starting to go.

226
00:17:29,000 --> 00:17:37,000
I'm just kidding.
Gets them every time.

227
00:17:37,000 --> 00:17:45,000
There really was no bacterium in
there of any kind.

228
00:17:45,000 --> 00:17:52,000
I know.  OK.  But we're still going
to go through this example.

229
00:17:52,000 --> 00:18:00,000
So we want to transfer this gene
into this organism.

230
00:18:00,000 --> 00:18:04,000
What are we going to do?
Well, the first thing we need to

231
00:18:04,000 --> 00:18:08,000
worry about is the fact that this
gene, the T gene here,

232
00:18:08,000 --> 00:18:12,000
is contained on a very large piece
of DNA.  As I said,

233
00:18:12,000 --> 00:18:16,000
it's got a thousand other genes.
It's about 4 million base pairs

234
00:18:16,000 --> 00:18:20,000
long.  And I'm only interested in
this one, so I need a way to isolate

235
00:18:20,000 --> 00:18:24,000
the T gene away from the other genes
present in this genome.

236
00:18:24,000 --> 00:18:28,000
So for this purpose I need a tool.
And the tool that I need is a

237
00:18:28,000 --> 00:18:41,000
restriction enzyme

238
00:18:41,000 --> 00:18:44,000
Restriction enzymes are nicely
described in your book.

239
00:18:44,000 --> 00:18:48,000
They are enzymes present in
bacterial cells which are designed

240
00:18:48,000 --> 00:18:51,000
to cut, to cleave DNA sequences.
And they do so in a site-specific

241
00:18:51,000 --> 00:18:55,000
fashion.  They are sequence-specific
--

242
00:18:55,000 --> 00:19:03,000
-- DNA cutting enzymes.

243
00:19:03,000 --> 00:19:09,000
We call them endonucleases,
but you don't have to worry about

244
00:19:09,000 --> 00:19:15,000
that term.  They are DNA cutting
enzymes.  Some of them produce,

245
00:19:15,000 --> 00:19:20,000
as you'll see in a moment, what we
call sticky ends,

246
00:19:20,000 --> 00:19:26,000
based on exactly how they cut the
DNA.  And others produce what we

247
00:19:26,000 --> 00:19:32,000
call blunt ends because they cut the
DNA in a slightly different way.

248
00:19:32,000 --> 00:19:35,000
There are hundreds of restriction
enzymes that have been purified from

249
00:19:35,000 --> 00:19:39,000
various bacteria.
There are now companies that will

250
00:19:39,000 --> 00:19:42,000
sell you the purified restriction
endonuclease.  There are whole

251
00:19:42,000 --> 00:19:46,000
businesses that are geared around
selling this stuff to molecular

252
00:19:46,000 --> 00:19:49,000
biologists around the world.
So you can order it up like you

253
00:19:49,000 --> 00:19:53,000
would order up a chemical from a
chemical supply house.

254
00:19:53,000 --> 00:19:57,000
As I said, these are site-specific,
sequence-specific DNA cutting

255
00:19:57,000 --> 00:20:01,000
enzymes.
So they recognize particular

256
00:20:01,000 --> 00:20:05,000
sequences in the DNA.
One such enzyme, which was one of

257
00:20:05,000 --> 00:20:09,000
the first discovered,
an enzyme called EcoR1,

258
00:20:09,000 --> 00:20:13,000
which comes from E. coli,
that's why it's EcoR1, R1 is

259
00:20:13,000 --> 00:20:17,000
probably restriction enzyme number
one, recognizes a particular

260
00:20:17,000 --> 00:20:21,000
sequence reading from the 5 prime
end of the DNA molecule towards the

261
00:20:21,000 --> 00:20:25,000
3 prime end.  It recognizes the
sequence G-A-A-T-T-C.

262
00:20:25,000 --> 00:20:29,000
And then the other side of that,
of course, is a 3 prime end.

263
00:20:29,000 --> 00:20:33,000
This is the polarity of DNA that
you've been shown in many cases

264
00:20:33,000 --> 00:20:37,000
before.  Now what is the reverse
strand?  What does the opposite,

265
00:20:37,000 --> 00:20:41,000
the complimentary strand of this
sequence look like?

266
00:20:41,000 --> 00:20:45,000
Can anybody tell me?
You want to yell it out in unison.

267
00:20:45,000 --> 00:20:52,000
Here is a G and --

268
00:20:52,000 --> 00:20:57,000
That was very good.
Notice anything about this?

269
00:20:57,000 --> 00:21:02,000
Yes.  It reads the same ways
forwards as backwards.

270
00:21:02,000 --> 00:21:05,000
G-A-A-T-T-C.  G-A-A-T-T-C.
What do we call that?  It's a

271
00:21:05,000 --> 00:21:09,000
palindrome.  Your book uses the word
MOM, which is a fairly boring

272
00:21:09,000 --> 00:21:13,000
palindrome.  It is reads the same
way forwards as backwards.

273
00:21:13,000 --> 00:21:16,000
I always like "A man, a plan,
a canal, Panama."  That's a

274
00:21:16,000 --> 00:21:20,000
palindrome, too.
It reads the same way forwards as

275
00:21:20,000 --> 00:21:24,000
backwards.  Anyway,
many, not all, but many restriction

276
00:21:24,000 --> 00:21:28,000
enzymes recognize palindromic
sequences.  They read the same way

277
00:21:28,000 --> 00:21:32,000
forwards as backwards.
OK?  And because of that they cleave

278
00:21:32,000 --> 00:21:37,000
the DNA on both strands at the same
position.  EcoR1,

279
00:21:37,000 --> 00:21:43,000
this enzyme that recognizes this
sequence will cleave the DNA between

280
00:21:43,000 --> 00:21:48,000
this phosphodiester bond,
between the phosphodiester bond

281
00:21:48,000 --> 00:21:53,000
between the G and the A.
And likewise on this strand.

282
00:21:53,000 --> 00:21:58,000
And the consequence of that is to
produce a break in the DNA,

283
00:21:58,000 --> 00:22:04,000
so you now have 5 prime G, 3 prime
C-T-A-A.

284
00:22:04,000 --> 00:22:07,000
Breaks occur here and the strands
get separated.

285
00:22:07,000 --> 00:22:10,000
They get pulled apart.
The hydrogen bonds that are holding

286
00:22:10,000 --> 00:22:14,000
these base pairs together are not
strong enough to hold the two

287
00:22:14,000 --> 00:22:17,000
molecules together,
at least most of the time,

288
00:22:17,000 --> 00:22:21,000
so most of the time they pull apart.
So you end up with a fragment that

289
00:22:21,000 --> 00:22:24,000
has an end that looks like this and
another fragment that has an end

290
00:22:24,000 --> 00:22:28,000
that looks like this,
A-A-T-C, here's the 3 prime end,

291
00:22:28,000 --> 00:22:32,000
G, and here's the 5 prime end.
OK?  So you snip it at either side

292
00:22:32,000 --> 00:22:36,000
and pull it apart.
Oops.  I did something wrong.

293
00:22:36,000 --> 00:22:41,000
I missed a T, yup, on both of them.
Thank you.  I don't think I've ever

294
00:22:41,000 --> 00:22:45,000
drawn those right.
In all the years I've taught this

295
00:22:45,000 --> 00:22:50,000
course, I always make it.
Not the same mistake necessarily

296
00:22:50,000 --> 00:22:54,000
but a mistake.
OK, so they make a break.

297
00:22:54,000 --> 00:22:59,000
And that then cleaves the DNA into
two pieces.

298
00:22:59,000 --> 00:23:04,000
Now, importantly,
at low enough temperatures these

299
00:23:04,000 --> 00:23:09,000
ends can find each other.
And we call them sticky because

300
00:23:09,000 --> 00:23:14,000
they are complimentary to one
another.  They can actually reform

301
00:23:14,000 --> 00:23:20,000
those base pairs.
So you would get a molecule which

302
00:23:20,000 --> 00:23:25,000
has 5 prime G,
three prime C-T-T-A-A.

303
00:23:25,000 --> 00:23:30,000
And then base paired to this
T-T-A-A would be an A-A-T-T sequence,

304
00:23:30,000 --> 00:23:36,000
which was covalently bound
on this side to the C.

305
00:23:36,000 --> 00:23:41,000
No.  Yeah, covalently bound on this
side to the C and non-covalently

306
00:23:41,000 --> 00:23:47,000
bound to this G.
OK?  So there are non-covalent

307
00:23:47,000 --> 00:23:53,000
bonds here and here and there are
base pairs in the middle that are

308
00:23:53,000 --> 00:23:59,000
holding these strands together.
We call this annealing.

309
00:23:59,000 --> 00:24:03,000
And, again, at low enough
temperatures you can get sticky ends

310
00:24:03,000 --> 00:24:07,000
of restriction enzyme cleaved sites
to come together this way.

311
00:24:07,000 --> 00:24:11,000
Now, importantly another enzyme
which we could use,

312
00:24:11,000 --> 00:24:16,000
discovered more or less the same
time, is an enzyme called DNA ligase,

313
00:24:16,000 --> 00:24:20,000
which you would have heard about in
the replication lectures.

314
00:24:20,000 --> 00:24:24,000
And DNA ligase will tie to
non-covalently linked pieces of DNA

315
00:24:24,000 --> 00:24:29,000
together by a covalent bond.
So DNA ligase will reintroduce this

316
00:24:29,000 --> 00:24:35,000
phosphodiester bond and this
phosphodiester bond to produce now a

317
00:24:35,000 --> 00:24:40,000
completely covalently-bound sequence.
OK?  So we can cut the DNA at sites

318
00:24:40,000 --> 00:24:46,000
we want to.  If we change the
temperature we can get these DNA

319
00:24:46,000 --> 00:24:51,000
molecules to come together in a
non-covalent fashion.

320
00:24:51,000 --> 00:24:57,000
And then, if we add this enzyme DNA
ligase, they will become covalently

321
00:24:57,000 --> 00:25:02,000
bond once again.  OK?
Key enzymes in our tool kit.

322
00:25:02,000 --> 00:25:06,000
As I said, there are other enzymes
that will cut DNA in a blunt fashion.

323
00:25:06,000 --> 00:25:10,000
They will not produce these sticky
ends.  I won't show you a new

324
00:25:10,000 --> 00:25:14,000
example of that today,
and it's not terribly important that

325
00:25:14,000 --> 00:25:18,000
you know about them,
but you can imagine that an enzyme

326
00:25:18,000 --> 00:25:22,000
that cuts in the middle between the
central positions of the recognition

327
00:25:22,000 --> 00:25:27,000
sequence will make a blunt end.
They won't be sticky.

328
00:25:27,000 --> 00:25:32,000
And those aren't as useful to us
because the sticky ends actually

329
00:25:32,000 --> 00:25:38,000
help us get two pieces of DNA
together in order to promote the

330
00:25:38,000 --> 00:25:43,000
cloning step at the end.
OK.  So the S. pyogenes genome that

331
00:25:43,000 --> 00:25:49,000
we need to isolate our T gene from
has a very large sequence.

332
00:25:49,000 --> 00:25:54,000
It's 4.1 times ten to the sixth
base pairs long all the way around

333
00:25:54,000 --> 00:26:00,000
the circle.  And I
was wondering. --

334
00:26:00,000 --> 00:26:10,000
Well, let me just remind you that
EcoR1 recognizes a 6 base pair

335
00:26:10,000 --> 00:26:20,000
recognition site.
So how many EcoR1 sites are there

336
00:26:20,000 --> 00:26:30,000
in this genome?
What's the frequency of EcoR1 sites

337
00:26:30,000 --> 00:26:37,000
along a piece of DNA?
Well, it has to be this particular

338
00:26:37,000 --> 00:26:42,000
sequence, G-A-A-T-C-C,
reading in that direction.

339
00:26:42,000 --> 00:26:47,000
The frequency of any given
nucleotide at any given site is one

340
00:26:47,000 --> 00:26:51,000
in four because there are four
nucleotides.  The frequency of six

341
00:26:51,000 --> 00:26:56,000
of a given sequence in a row is one
over four to the sixth,

342
00:26:56,000 --> 00:27:01,000
which is one in 4,096.
So every 4,000 base pairs or so,

343
00:27:01,000 --> 00:27:05,000
at random, you will find an EcoR1
site.  OK?  They won't be equally

344
00:27:05,000 --> 00:27:09,000
distributed.  There will be some
parts of the genome where  there are

345
00:27:09,000 --> 00:27:13,000
many by chance,
other parts where there are a few,

346
00:27:13,000 --> 00:27:18,000
but on average there will be about
one every 4,000 base pairs.

347
00:27:18,000 --> 00:27:22,000
And since the genome is 4 million
base pairs long that means that

348
00:27:22,000 --> 00:27:26,000
there's going to be about a thousand
EcoR1 sites scattered around

349
00:27:26,000 --> 00:27:31,000
this genome.  OK?
And they might be close together in

350
00:27:31,000 --> 00:27:35,000
some places, further apart in other
places, and so on around the gene.

351
00:27:35,000 --> 00:27:40,000
Now, the T gene is in a particular
place within the genome.

352
00:27:40,000 --> 00:27:44,000
I'm going to draw it blue.
Here's the T gene.  And when

353
00:27:44,000 --> 00:27:48,000
deciding what restriction
endonuclease, what restriction

354
00:27:48,000 --> 00:27:53,000
enzyme to use to cleave the T gene
in an intact fashion,

355
00:27:53,000 --> 00:27:57,000
I'll consult the sequence,
which I know, I'll choose an enzyme

356
00:27:57,000 --> 00:28:02,000
which I know won't cleave this gene,
won't cut this gene.

357
00:28:02,000 --> 00:28:06,000
There are no EcoR1 sites within this
gene.  OK?  So there might be one on

358
00:28:06,000 --> 00:28:11,000
this side and there might be one on
this side, but I've chosen EcoR1

359
00:28:11,000 --> 00:28:16,000
because I know there are no
G-A-A-T-T-C sequences within the T

360
00:28:16,000 --> 00:28:21,000
gene.  OK?  So to give you a visual
depiction of what we're talking

361
00:28:21,000 --> 00:28:26,000
about, more visual aids,
I brought with me a very,

362
00:28:26,000 --> 00:28:31,000
very dangerous piece of rope,
a flesh eating piece of rope.

363
00:28:31,000 --> 00:28:34,000
They never fall for it twice,
do they?  In which I've depicted the

364
00:28:34,000 --> 00:28:38,000
S. pyogenes genome as a piece of
covalently bound rope here.

365
00:28:38,000 --> 00:28:42,000
And the EcoR1 sites are shown in
yellow.  OK?  That's really what

366
00:28:42,000 --> 00:28:46,000
they are.  They're just little tags
basically that the specific enzymes

367
00:28:46,000 --> 00:28:50,000
recognize.  They're not there for
the purposes of the enzyme

368
00:28:50,000 --> 00:28:54,000
recognizing them.
They're just there.

369
00:28:54,000 --> 00:28:58,000
And the enzyme can recognize them.
OK?  And here I've colored in blue

370
00:28:58,000 --> 00:29:01,000
the T gene.
And you'll see that there are no

371
00:29:01,000 --> 00:29:05,000
EcoR1 sites within the T gene.
So now if I take this purified

372
00:29:05,000 --> 00:29:09,000
piece of DNA that I recovered from S.
pyogenes and I put in solution with

373
00:29:09,000 --> 00:29:13,000
the restriction enzyme EcoR1 in the
right sort of buffer and so on,

374
00:29:13,000 --> 00:29:16,000
the restriction enzyme will do what
it's able to do.

375
00:29:16,000 --> 00:29:20,000
It will go along and find those
sites and cut them.

376
00:29:20,000 --> 00:29:24,000
OK?  Everywhere it sees one it will
cut one.  You know what I really

377
00:29:24,000 --> 00:29:28,000
should do?  I should figure out how
to put the rope back together.

378
00:29:28,000 --> 00:29:32,000
That would be a trick.
I don't know how to do that.

379
00:29:32,000 --> 00:29:37,000
I should work on that.  That would
be great.  I should do that.

380
00:29:37,000 --> 00:29:41,000
All right.  So we've cleaved our
DNA molecule into fragments.

381
00:29:41,000 --> 00:29:46,000
OK?  And the S. pyogenes genome
that we drew up on the board will

382
00:29:46,000 --> 00:29:51,000
likewise be separated into fragments.
It will be separated into about a

383
00:29:51,000 --> 00:29:56,000
thousand fragments because there are
about a thousand sites.

384
00:29:56,000 --> 00:30:01,000
OK?  I now want to separate those
fragments from one another.

385
00:30:01,000 --> 00:30:05,000
In order to clone the T gene,
I need to separate the T gene away

386
00:30:05,000 --> 00:30:09,000
from all the other genes and all the
other DNA sequences in this genome.

387
00:30:09,000 --> 00:30:13,000
So how do I do it?  Well, I take
advantage of the fact that the

388
00:30:13,000 --> 00:30:17,000
pieces of DNA that I've liberated
are different lengths.

389
00:30:17,000 --> 00:30:21,000
You can see that there are some
short ones, there are some long ones,

390
00:30:21,000 --> 00:30:25,000
and it's actually quite easy to
separate DNA based on its size.

391
00:30:25,000 --> 00:30:29,000
The way you do that is to introduce
the DNA solution that contains the

392
00:30:29,000 --> 00:30:34,000
fragments of all sizes into what we
call an agarose gel.

393
00:30:34,000 --> 00:30:39,000
It's like a slab of Jell-O.
At one end, as shown here, here's

394
00:30:39,000 --> 00:30:44,000
the slab of Jell-O in which little
wells have been cut out,

395
00:30:44,000 --> 00:30:48,000
little indentations have been placed,
we introduce the DNA solution,

396
00:30:48,000 --> 00:30:53,000
usually with a colored dye to let us
know what we're doing,

397
00:30:53,000 --> 00:30:58,000
and then we put buffer on top of the
gel and at the two wells at the end

398
00:30:58,000 --> 00:31:03,000
of the gel, and then we apply
an electric field.

399
00:31:03,000 --> 00:31:07,000
This is called gel electrophorisis.
We apply a positive charge on one

400
00:31:07,000 --> 00:31:11,000
end, a negative charge on the other
end.  Which way will the DNA go?

401
00:31:11,000 --> 00:31:15,000
Which way will the DNA go?  Is DNA
positively charges or negatively

402
00:31:15,000 --> 00:31:19,000
charged?  It's negatively charged
because all those phosphates which

403
00:31:19,000 --> 00:31:23,000
carry a negative charge.
So in neutral pH buffer it will

404
00:31:23,000 --> 00:31:27,000
move towards the positive pole.
And so the DNA will actually

405
00:31:27,000 --> 00:31:31,000
separate.
And that's what you're seeing here.

406
00:31:31,000 --> 00:31:35,000
These are gels of different
concentrations of the stuff that

407
00:31:35,000 --> 00:31:39,000
makes them stiff.
So at a low percentage you see this

408
00:31:39,000 --> 00:31:42,000
fragmentation of fragments.
The larger fragments don't go very

409
00:31:42,000 --> 00:31:46,000
far, the smaller fragments go
further into the gel,

410
00:31:46,000 --> 00:31:49,000
and at different concentrations of
this stuff you'll get different

411
00:31:49,000 --> 00:31:53,000
separations in different positions.
And so with this separation

412
00:31:53,000 --> 00:31:57,000
technique I can isolate this
fragment, which is of a particular

413
00:31:57,000 --> 00:32:00,000
size, away from other fragments.
So if you imagine this is the gel

414
00:32:00,000 --> 00:32:03,000
here, I put a negative charge here,
a positive charge here, the

415
00:32:03,000 --> 00:32:07,000
different DNA fragments are going to
separate along the gel with the long

416
00:32:07,000 --> 00:32:10,000
ones at the top and the short ones
at the bottom,

417
00:32:10,000 --> 00:32:13,000
and the middle sized ones will
separate in the middle and the T

418
00:32:13,000 --> 00:32:17,000
gene will separate right there.
And, if I have a lot of DNA to

419
00:32:17,000 --> 00:32:20,000
start with, I'm going to have a lot
of molecules of the T gene in this

420
00:32:20,000 --> 00:32:23,000
position on the gel.
And if I shine a UV light,

421
00:32:23,000 --> 00:32:27,000
which was what was done here,
I can literally see that position.

422
00:32:27,000 --> 00:32:31,000
And since I know how big it is,
because I know the sequence of this

423
00:32:31,000 --> 00:32:35,000
organism, I can know that it's
exactly 1,512 base pairs long.

424
00:32:35,000 --> 00:32:39,000
And, therefore, it should run right
there in this particular position on

425
00:32:39,000 --> 00:32:43,000
the gel.  So I can purify it.
Now, usually when I purify it,

426
00:32:43,000 --> 00:32:47,000
I purify a bunch of other things
that are of similar size.

427
00:32:47,000 --> 00:32:51,000
So I don't usually get it all only
this molecule,

428
00:32:51,000 --> 00:32:55,000
but I do a pretty good job of
purifying it.  And now I need to

429
00:32:55,000 --> 00:32:59,000
amplify it.  I've got some of that
DNA, but I need to have

430
00:32:59,000 --> 00:33:03,000
more of the DNA.
I need to have much,

431
00:33:03,000 --> 00:33:07,000
much, many, many more copies in
order to be able to do the

432
00:33:07,000 --> 00:33:12,000
manipulations I want to do
downstream.  So the next thing I

433
00:33:12,000 --> 00:33:16,000
need to do is get this piece of DNA,
and the other ones that I'm less

434
00:33:16,000 --> 00:33:21,000
interested in,
into bacteria and get the bacteria

435
00:33:21,000 --> 00:33:25,000
to make more and more of it.
So what I do in principle is to

436
00:33:25,000 --> 00:33:30,000
introduce the DNA into
different bacteria.

437
00:33:30,000 --> 00:33:35,000
There you go.  And now I need those
bacteria to make more of that stuff

438
00:33:35,000 --> 00:33:41,000
in order to give me what I need.
But there are certain problems.

439
00:33:41,000 --> 00:33:46,000
There are certain problems
associated with what I've just told

440
00:33:46,000 --> 00:33:52,000
to you.  The problems are that I
cannot tell which of these folks got

441
00:33:52,000 --> 00:33:57,000
the right gene.
That's one problem.

442
00:33:57,000 --> 00:34:03,000
I need to figure out a way to know
who got the T gene and who didn't.

443
00:34:03,000 --> 00:34:07,000
I also need to know who got any
piece of DNA versus who got nothing.

444
00:34:07,000 --> 00:34:12,000
The rest of you didn't get any DNA.
You are of no interest to me.

445
00:34:12,000 --> 00:34:16,000
If you were bacteria, I wish you
would die.  It's actually true.

446
00:34:16,000 --> 00:34:21,000
You'll see in a second.  The rest
of these guys I'm somewhat

447
00:34:21,000 --> 00:34:26,000
interested in,
and I'm really interested in him

448
00:34:26,000 --> 00:34:30,000
because he's got the TG.
I've picked on you a lot,

449
00:34:30,000 --> 00:34:34,000
haven't I?
So those are two of the problems.

450
00:34:34,000 --> 00:34:38,000
And the third problem is that I
need these guys to divide,

451
00:34:38,000 --> 00:34:41,000
which they're not terribly willing
to do.  I need them to divide,

452
00:34:41,000 --> 00:34:44,000
but bacteria will divide.  But the
bacteria, if these guys were

453
00:34:44,000 --> 00:34:48,000
bacteria, wouldn't know what to do
with that little fragment of DNA

454
00:34:48,000 --> 00:34:51,000
that I gave them.
They wouldn't know how to replicate

455
00:34:51,000 --> 00:34:54,000
it, how to make more if it because
I've just given them a naked piece

456
00:34:54,000 --> 00:34:58,000
of DNA and that's not terribly
useful.  So we need

457
00:34:58,000 --> 00:35:04,000
to overcome that.
And I'm going to tell you how.

458
00:35:04,000 --> 00:35:13,000
The process that we just described,
the introduction of DNA into the

459
00:35:13,000 --> 00:35:22,000
bacterium is called transformation,
bacterial transformation.  Again,

460
00:35:22,000 --> 00:35:31,000
here's a bacterial cell
with its own genome.

461
00:35:31,000 --> 00:35:35,000
We treat the bacterium with
chemicals that sort of loosen up the

462
00:35:35,000 --> 00:35:39,000
membrane a little bit or we
electroshock the bacteria which

463
00:35:39,000 --> 00:35:43,000
blows holes in the membrane.
And then when there are holes in

464
00:35:43,000 --> 00:35:47,000
the membrane, these little fragments
of DNA which I'm representing here,

465
00:35:47,000 --> 00:35:51,000
maybe I'll just show it as a single
linear piece of DNA,

466
00:35:51,000 --> 00:35:55,000
can float in inside the cytoplasm of
the bacteria.  They literally just

467
00:35:55,000 --> 00:35:59,000
float in there.  OK?
Now, the bacterium,

468
00:35:59,000 --> 00:36:04,000
if they knew what to do with this
piece of DNA, are remarkably good

469
00:36:04,000 --> 00:36:09,000
biofactories because bacteria will
divide at optimal conditions three

470
00:36:09,000 --> 00:36:14,000
times every hour,
three divisions every hour,

471
00:36:14,000 --> 00:36:20,000
which means 72 divisions every day.

472
00:36:20,000 --> 00:36:25,000
And if they could duplicate this DNA
every time they divided,

473
00:36:25,000 --> 00:36:30,000
if they could and they were able to
divide 72 times in a day,

474
00:36:30,000 --> 00:36:35,000
if there were one copy of that piece
of DNA per cell at the beginning of

475
00:36:35,000 --> 00:36:41,000
the process, just one cell that had
that piece of DNA in it then at the

476
00:36:41,000 --> 00:36:46,000
end of the day there could be two to
the seventy-second molecules of DNA.

477
00:36:46,000 --> 00:36:51,000
OK?  72 divisions.  Two to the
seventy-second which is roughly

478
00:36:51,000 --> 00:36:57,000
equivalent to ten to the twentieth
copies.  Now, each of you has ten to

479
00:36:57,000 --> 00:37:02,000
the thirteenth cells in you.
So you've got ten to the thirteenth

480
00:37:02,000 --> 00:37:06,000
or so copies of any particular piece
of DNA.  This is ten to the seventh

481
00:37:06,000 --> 00:37:10,000
times more than that,
so that's 10 million persons' worth

482
00:37:10,000 --> 00:37:14,000
of a given piece of DNA.
It's really quite remarkable.

483
00:37:14,000 --> 00:37:18,000
Now, Claudette always makes me
point out of the fact that you never

484
00:37:18,000 --> 00:37:22,000
reach this theoretical limit because
you would need an MIT's worth of

485
00:37:22,000 --> 00:37:26,000
bacterial solution to do it.
So in practice, when we do this

486
00:37:26,000 --> 00:37:30,000
sort of experiment,
we get about ten to the tenth to ten

487
00:37:30,000 --> 00:37:34,000
to the thirteenth bacteria
at the end.

488
00:37:34,000 --> 00:37:37,000
And, if each bacteria were able to
copy this DNA faithfully,

489
00:37:37,000 --> 00:37:41,000
we would have about ten to the tenth
or ten to the thirteenth copies of

490
00:37:41,000 --> 00:37:45,000
the DNA.  Still quite impressive for
not a lot of work.

491
00:37:45,000 --> 00:37:48,000
The problem I've already alluded to
is that the bacteria don't know how

492
00:37:48,000 --> 00:37:52,000
to deal with this introduced piece
of DNA.  This piece of DNA,

493
00:37:52,000 --> 00:37:56,000
for it to be brought up to ten to
the tenth or ten to the thirteenth

494
00:37:56,000 --> 00:38:00,000
copies needs to be replicated.
The DNA needs to be replicated.

495
00:38:00,000 --> 00:38:16,000
What do we know about replication?

496
00:38:16,000 --> 00:38:20,000
What did you learn about
replication?  What is the key thing

497
00:38:20,000 --> 00:38:23,000
that a piece of DNA needs in order
for it to be replicated?

498
00:38:23,000 --> 00:38:26,000
An origin of replication,
right?  And these fragments most

499
00:38:26,000 --> 00:38:30,000
likely are not carry an origin of
replication, so they're not going to

500
00:38:30,000 --> 00:38:34,000
be replicated.
So for this purpose we need an

501
00:38:34,000 --> 00:38:38,000
origin of replication.
And you'll see how we accomplish

502
00:38:38,000 --> 00:38:42,000
that.  We also mentioned the fact
that most of the bacteria,

503
00:38:42,000 --> 00:38:46,000
when we do this transformation,
this is actually a relatively

504
00:38:46,000 --> 00:38:50,000
inefficient process.
So most bacteria get no DNA.

505
00:38:50,000 --> 00:38:54,000
And they are, of course, not
interesting to us.

506
00:38:54,000 --> 00:38:58,000
That's all of you guys in the back
there.  So we need to get

507
00:38:58,000 --> 00:39:03,000
rid of all of you.
And then we need to find which

508
00:39:03,000 --> 00:39:09,000
bacterium has the clone of interest.
Which bacterium has the piece of

509
00:39:09,000 --> 00:39:15,000
DNA of interest?
This guy compared to the rest of

510
00:39:15,000 --> 00:39:21,000
them.  So the solutions to all of
those problems come in the form of

511
00:39:21,000 --> 00:39:27,000
what we call vectors.
The piece of DNA that we introduce

512
00:39:27,000 --> 00:39:33,000
at the beginning is not an isolated
fragment of DNA, in fact.

513
00:39:33,000 --> 00:39:49,000
Instead, we introduce the piece of

514
00:39:49,000 --> 00:39:56,000
DNA in the context of another DNA
molecule called a vector.

515
00:39:56,000 --> 00:40:03,000
Vectors, for the most part,
are derived from naturally occurring

516
00:40:03,000 --> 00:40:10,000
small DNA molecules that are carried
in many bacteria outside of the main

517
00:40:10,000 --> 00:40:16,000
bacterial genome.
This is the main bacterial genome.

518
00:40:16,000 --> 00:40:22,000
Many bacteria carry within their
cytoplasm small copies of DNA,

519
00:40:22,000 --> 00:40:27,000
also circular, called plasmids.
And these are short.  They might be

520
00:40:27,000 --> 00:40:33,000
on average 5,000 base pairs long
compared to a couple of million base

521
00:40:33,000 --> 00:40:39,000
pairs long.  So they're small
circular pieces of DNA.

522
00:40:39,000 --> 00:40:43,000
They get transferred from bacterium
to bacterium in the wild for the

523
00:40:43,000 --> 00:40:47,000
purposes of genetic exchange between
bacteria, also for transmitting drug

524
00:40:47,000 --> 00:40:51,000
resistance or pathogenic properties
from one bacteria to another.

525
00:40:51,000 --> 00:40:55,000
So they do get exchanged from
bacteria to bacteria.

526
00:40:55,000 --> 00:40:59,000
They were discovered a long time
ago and it was recognized that they

527
00:40:59,000 --> 00:41:03,000
would be very useful as vehicle,
vectors for cloned DNA.

528
00:41:03,000 --> 00:41:07,000
They have three key features,
which I will illustrate in this

529
00:41:07,000 --> 00:41:12,000
diagram.  One is the origin of
replication.  So they do replicate

530
00:41:12,000 --> 00:41:16,000
inside bacterial cells.
They have their own origin of

531
00:41:16,000 --> 00:41:21,000
replication.  So you can see where
we're going here.

532
00:41:21,000 --> 00:41:26,000
We're going to put our piece of DNA,
along with this,

533
00:41:26,000 --> 00:41:31,000
in order to get the replication of
our DNA as well.

534
00:41:31,000 --> 00:41:35,000
They have restriction sites.
Of course, they're going to have

535
00:41:35,000 --> 00:41:40,000
restriction sites throughout their
DNA sequence.  But they're designed

536
00:41:40,000 --> 00:41:45,000
now on purpose to have particular
restriction sites in a particular

537
00:41:45,000 --> 00:41:50,000
region of the piece of DNA which we
use for cloning purposes.

538
00:41:50,000 --> 00:41:55,000
So there might be EcoR1 site here
and nowhere else in this plasmid,

539
00:41:55,000 --> 00:42:00,000
another enzyme represented once, and
so on and so forth.

540
00:42:00,000 --> 00:42:05,000
Because we're going to introduce our
DNA into here and we don't want to

541
00:42:05,000 --> 00:42:10,000
disrupt the sequence of the rest of
the plasmid.  And the final thing

542
00:42:10,000 --> 00:42:15,000
they have of value is a drug
resistance gene,

543
00:42:15,000 --> 00:42:20,000
sometimes multiple drug resistance
genes.  An example of a drug

544
00:42:20,000 --> 00:42:25,000
resistance gene is the amp
resistance gene,

545
00:42:25,000 --> 00:42:30,000
and this confers resistance to a
common antibiotic called ampicillin,

546
00:42:30,000 --> 00:42:35,000
very similar to penicillin.
If you treat most bacteria with

547
00:42:35,000 --> 00:42:39,000
penicillin they will die,
with ampicillin, sorry, they will

548
00:42:39,000 --> 00:42:43,000
die.  If you treat a bacterium
that's carrying a plasmid that has

549
00:42:43,000 --> 00:42:47,000
an ampicillin resistance gene the
bacterium will live,

550
00:42:47,000 --> 00:42:51,000
OK, because it inactivates the
ampicillin.  It makes it non-toxic

551
00:42:51,000 --> 00:42:55,000
to the bacteria.
And now you can see how we're going

552
00:42:55,000 --> 00:42:59,000
to distinguish bacteria that get DNA
from bacteria that don't

553
00:42:59,000 --> 00:43:04,000
get any DNA.
The bacteria that get DNA are going

554
00:43:04,000 --> 00:43:09,000
to get a drug resistance gene.
So they're going to live in the

555
00:43:09,000 --> 00:43:14,000
presence of ampicillin.
If you don't get any DNA and you

556
00:43:14,000 --> 00:43:20,000
get treated with ampicillin you're
going to die.  OK.  So, quickly.

557
00:43:20,000 --> 00:43:36,000
I've taken my S.

558
00:43:36,000 --> 00:43:40,000
pyogenes genome and I cut it up into
EcoR1 sites.  I've cut it with EcoR1,

559
00:43:40,000 --> 00:43:44,000
so I've cut it into EcoR1 fragments.
And now I'm representing them as

560
00:43:44,000 --> 00:43:48,000
double-stranded with their sticky
ends showing.  Here's the end of an

561
00:43:48,000 --> 00:43:52,000
EcoR1 site.  Here's the end of an
EcoR1 site.  I'm just going to show

562
00:43:52,000 --> 00:43:56,000
you two for the sake of simplicity.
Here's another fragment liberated

563
00:43:56,000 --> 00:44:00,000
by EcoR1 from the S.
pyogenes genome.

564
00:44:00,000 --> 00:44:06,000
Maybe these are two of similar size
and I purified them together when I

565
00:44:06,000 --> 00:44:12,000
used my agarose gel technique.
So these are two pieces of DNA.

566
00:44:12,000 --> 00:44:18,000
And this one contains our friend
the T gene.  We've liberated these,

567
00:44:18,000 --> 00:44:24,000
they have their sticky ends, and
then we mix them with a plasmid that

568
00:44:24,000 --> 00:44:31,000
likewise has been cleaved
with EcoR1.

569
00:44:31,000 --> 00:44:36,000
So we have lots and lots of cut
plasmid that has the complimentary

570
00:44:36,000 --> 00:44:41,000
sticky ends.  OK?
This plasmid is that thing.

571
00:44:41,000 --> 00:44:46,000
I'm showing it as double-stranded
now so I can emphasize the sticky

572
00:44:46,000 --> 00:44:51,000
ends.  When I lower the temperature,
at some frequency this DNA molecule

573
00:44:51,000 --> 00:44:56,000
with find its way here,
an anneal at this end over here,

574
00:44:56,000 --> 00:45:01,000
at this end over here, and this
piece of DNA will find its way to a

575
00:45:01,000 --> 00:45:07,000
different plasmid an anneal over
here and over here.

576
00:45:07,000 --> 00:45:12,000
If I wait a while for that annealing
process to take place and then I add

577
00:45:12,000 --> 00:45:18,000
DNA ligase, the enzyme that seals
the nicks that form when those

578
00:45:18,000 --> 00:45:24,000
things come together,
I will get fully closed

579
00:45:24,000 --> 00:45:30,000
double-stranded DNA molecules which
are the plasmid plus

580
00:45:30,000 --> 00:45:35,000
a new piece of DNA.
This one will give some chunk of the

581
00:45:35,000 --> 00:45:40,000
S. pyogenes genome I don't care
about.  This one will give a plasmid

582
00:45:40,000 --> 00:45:45,000
that has a piece of the S.
pyogenes genome that carries the T

583
00:45:45,000 --> 00:45:50,000
gene.  This one I do care about.
OK?  So, in the final minute.

584
00:45:50,000 --> 00:46:04,000
I now take these plasmids that I've

585
00:46:04,000 --> 00:46:10,000
generated by this technique,
these recombinant plasmids, and I

586
00:46:10,000 --> 00:46:16,000
introduce them into bacteria in
culture using this transformation

587
00:46:16,000 --> 00:46:22,000
technique.  And then I plate them
onto a Petri dish.

588
00:46:22,000 --> 00:46:28,000
And the Petri dish,
importantly, has agar for the

589
00:46:28,000 --> 00:46:35,000
bacterial colonies to grow and it
contains ampicillin.

590
00:46:35,000 --> 00:46:40,000
It contains the drug ampicillin.
Now, as I said, the transformation

591
00:46:40,000 --> 00:46:45,000
process is inefficient.
Most bacteria that I treat in the

592
00:46:45,000 --> 00:46:50,000
transformation process will get no
DNA.  They'll land on this plate.

593
00:46:50,000 --> 00:46:55,000
And what will happen to them?
They'll die.  The plasmids that did

594
00:46:55,000 --> 00:47:00,000
get DNA, sorry,
the bacterium that did pick up a

595
00:47:00,000 --> 00:47:05,000
plasmid will have picked up an
ampicillin resistance gene.

596
00:47:05,000 --> 00:47:09,000
They will grow.
And they grow on little colonies,

597
00:47:09,000 --> 00:47:13,000
into little colonies after about a
day which have about a million cells

598
00:47:13,000 --> 00:47:18,000
each.  And they're separated from
one another.  So this colony might

599
00:47:18,000 --> 00:47:22,000
have that plasmid in it.
Every cell in this colony has that

600
00:47:22,000 --> 00:47:26,000
plasmid in it.
And this colony might have that

601
00:47:26,000 --> 00:47:31,000
plasmid in it.  OK?
I'm interested in this one because I

602
00:47:31,000 --> 00:47:35,000
want to now purify large quantities
of that piece of DNA.

603
00:47:35,000 --> 00:47:40,000
I'm actually not interested in this
one.  And next lecture we'll talk

604
00:47:40,000 --> 00:47:44,000
about how you specifically purify
this, isolate and identify this

605
00:47:44,000 --> 00:47:47,000
colony compared to the rest.