PROFESSOR: Now having cut
DNA, we then need to
do something else.
What's the next thing
we have to do?
AUDIENCE: Paste it.
PROFESSOR: Paste it right.
Oh, I put past.
It should be paste.
There we go.
We should paste it.
So, how are we going
to paste our DNA?
So let's take some human DNA.
We'll take your human DNA.
We'll add EcoR1 to it.
And your human DNA is going to
get cut up in lots of little
pieces of length about 4,000.
And I'm writing R1 at the ends
because these pieces of DNA
have EcoR1 sites.
Now, I can take that human DNA
and I can combine it with
other pieces of DNA.
Here's a piece of DNA.
Remember it had this
overhang like that.
T-T-A-A was this piece
here of human DNA.
And I could take another piece
of DNA that matched it a
A-A-T-T.
And it doesn't have
to be human.
It could be something else.
It could be zebra.
Some human DNA, some zebra DNA,
and by that base pairing
of those four bases, they'll
sort of stick.
It's not that strong.
It's four bases of base pairing,
but they'll stick.
Now I'd like to glue
them together.
What's the word for attaching
together DNA strands?
We talked about it--
Ligate, we want to ligate
them together.
So now we go back to our MIT
engineers and say, please
invent me a protein that's
able to ligate
together pieces of DNA.
But actually, who invented
it first?
Bacteria invented it first.
And it's called?
Ligase.
So all we have to do
is add ligase.
So it was kind of useful
to know how
DNA replication worked.
We add ligase.
And what does ligase
do for a living?
For a living, ligase is paid to
go around and find pieces
of DNA that have nicks and seal
them back up by repairing
the sugar phosphate
backbone of DNA.
And again, ancient molecular
biologists would prove their
mettle by purifying ligase and
using it in their reactions.
And today, where do
we get ligase?
It's in the catalog.
Ligase is in the catalog,
reasonably cheap.
So, when we add ligase we could
now get a hybrid piece
of DNA that was half human
and half zebra.
I got to say, this freaks
some people out.
This doesn't freak me out
because it's just a sequence
of nucleotides.
If I give you a sequence of
nucleotides, to my mind,
there's nothing human
or zebra about it.
It's a chemical.
And you can make bonds between
chemicals and all that.
So, we now pasted our
DNA using ligase.
Ligase from the catalog.
Now the question is, what are we
going to paste our DNA to?
What do we paste our DNA to?
We're going to paste our DNA to
a fascinating other piece
of DNA like this.
I'll make that a little
bigger here.
That has an EcoR1 site,
has an EcoR1 site.
And we're going to take our
human DNA and paste it in such
a way that it makes a circle.
This piece of DNA is a vector.
Vector means it travels
around.
It carries things with it.
So I want this vector to be
able to replicate if I
transfer it to E Coli.
So that's the trick.
I would like to be able to
attachment my human DNA to
this vector DNA, and when I
transfer it to E coli, have
this thing be able to grow.
So that means we need to invent
a piece of DNA, a
vector, that is capable
of causing E. coli
to replicate itself.
It's got to have all the
instructions to cause E. coli
to replicate this
piece of DNA.
So this is another amazing
bit of engineering.
How do we invent just the right
sequence of letters that
would allow E. coli to
replicate this thing?
AUDIENCE: Already
been invented.
PROFESSOR: Sorry.
AUDIENCE: It's already been
invented by E. coli.
PROFESSOR: It's already
been invented by E.
coli, hasn't it?
So you're getting the theme
here, is that we don't
actually do anything in
molecular biology.
Now look, in fairness, life's
had 3.5 billion years.
We've been at this a decade
or two, on the
whole, maybe three.
On the whole, life's had
a lot more time to
work this stuff out.
And usually the best solution
is to look in nature to see
where nature has already
done it for you.
So E. coli, it turns out,
replicates its own chromosome.
You could use the machinery
from its own chromosome.
But it turns out even
better than that.
E. coli has the following.
Here's its big chromosome, four
million letters of E.
coli chromosome.
E. Coli also has within
it little circles
of DNA called plasmids.
These little circles
of DNA are able to
autonomously replicate.
They can copy themselves.
Or that is to say, E.
coli copies them.
These little plasmids have the
full replication instructions
encoded in them.
Now why does E. coli have
these plasmids?
What's in these plasmids?
What's going on?
Well let's blow up one
of these plasmids.
It's a big circle.
And it has an instruction here,
a sequence, called an
origin of replication - or
amongst friends, just ORI.
It's called ORI.
You'll find on maps, origin
of replication.
That sequence alone is enough to
cause E. coli to open it up
and start doing its DNA
replication from the origin.
But what tells you why this is
so important is that these
plasmids typically have
one or two genes.
And the one or two genes that
they typically have are genes
that encode a protein that gives
them resistance to an
antibiotic, like say,
penicillin.
There could be a gene that
gives you penicillin
resistance.
That's kind of cool.
E. coli, or other bacteria--
not E. coli necessarily
for penicillin--
carry around often
little circles.
And these little circles encode
genes that give them
resistance to streptomycin,
penicillin, ampicillin, all
sorts of things.
This allows it to grow, even if
you're taking penicillin.
Now, that's pretty clever.
How did E. Coli come up?
How is it so smart to know to
have come up with a gene able
to break down penicillin, given
that we've only been
using penicillin since
the 1940s?
Pretty fast for E. coli to have
figured out how to do
that, isn't it?
Oh yeah, who invented
penicillin?
Nature invented penicillin.
It's produced by fungi.
Fungi have been fighting E.
Coli with penicillin for
millions of years.
E. coli didn't invent
it for us.
E. Coli invented it because it's
in a war down there at
the single cell level
with fungi.
Fungi make antibi--
You see, we think
we're so cool.
We make antibiotics.
Well we've been making
antibiotics
only since the 1940s.
Nature's been making antibiotics
forever.
Just like the viruses are
infecting E. coli and E. coli
needs an immune system
against the viruses.
Well the fungi are throwing
antibiotics at the bacteria
and it needs a defense
mechanism.
And the defense mechanism are
these genes that can break
down those antibiotics.
And as usual, we just
come along and we
say, oh look at that.
It's already been invented.
Kind of cool.
We'll use that one too.
Now why are these on these
little circles?
They're on the little circles
because when this bacteria has
lived a long and happy life and
it dies and it spews its
guts out, neighboring bacteria
suck up the DNA.
Sounds a little cannibalistic
or something.
But that's how it
is down there at
the single cell level.
The neighboring bacteria
will suck up DNA.
And why is that cool?
It's cool because they can
acquire these resistances.
That's pretty impressive.
They can acquire
the resistance.
And in fact, it even works
across species.
That's why you don't put it
on the chromosome here.
Because E. coli could pick it up
from another species that's
not E. coli, but related
enough that it
can use that plasmid.
And you can transfer antibiotic
resistance across
many species of bacteria.
So the bacteria like to suck up
DNA and see what they find.
Pretty cool.
By the way, this is also why
indiscriminate use of
antibiotics, for example, in
animal feed, or when you were
given antibiotics by the MIT
health service and are told to
take them for two weeks and you
take them for five days,
you're not doing anything
very good.
You're just selecting for
bacteria that can grow in the
presence of antibiotics and
selecting for multidrug
resistance in the spread
of antibiotics.
We have to be pretty careful.
Because all these mechanisms
of swapping antibiotic
resistance are pretty
impressive tricks.
Anyway, so these things exist.
And originally, molecular
biologists
discovered these plasmids.
They purified these plasmids.
And the deal was this,
they cut the
plasmid with EcoR1, ideally.
So I'm going to put this
up here, vectors.
They cut the plasmid
with EcoR1.
They ligate human DNA into it.
And they get these circles.
And there you go.
Now today, obviously, if I
wanted to do this experiment,
would I purify the
plasmid myself?
No.
Where would I get the
plasmid from?
AUDIENCE: Catalog.
PROFESSOR: It's in
the catalog.
There's a whole section
a plasmids here.
They have vectors
of all sorts.
So you get it from
the catalog.
All right.
