WEBVTT

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PROFESSOR: Now having cut
DNA, we then need to

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do something else.

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What's the next thing
we have to do?

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AUDIENCE: Paste it.

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PROFESSOR: Paste it right.

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Oh, I put past.

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It should be paste.

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There we go.

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We should paste it.

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So, how are we going
to paste our DNA?

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So let's take some human DNA.

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We'll take your human DNA.

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We'll add EcoR1 to it.

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And your human DNA is going to
get cut up in lots of little

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pieces of length about 4,000.

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And I'm writing R1 at the ends
because these pieces of DNA

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have EcoR1 sites.

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Now, I can take that human DNA
and I can combine it with

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other pieces of DNA.

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Here's a piece of DNA.

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Remember it had this
overhang like that.

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T-T-A-A was this piece
here of human DNA.

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And I could take another piece
of DNA that matched it a

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A-A-T-T.

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And it doesn't have
to be human.

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It could be something else.

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It could be zebra.

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Some human DNA, some zebra DNA,
and by that base pairing

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of those four bases, they'll
sort of stick.

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It's not that strong.

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It's four bases of base pairing,
but they'll stick.

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Now I'd like to glue
them together.

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What's the word for attaching
together DNA strands?

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We talked about it--

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Ligate, we want to ligate
them together.

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So now we go back to our MIT
engineers and say, please

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invent me a protein that's
able to ligate

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together pieces of DNA.

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But actually, who invented
it first?

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Bacteria invented it first.

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And it's called?

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Ligase.

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So all we have to do
is add ligase.

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So it was kind of useful
to know how

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DNA replication worked.

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We add ligase.

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And what does ligase
do for a living?

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For a living, ligase is paid to
go around and find pieces

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of DNA that have nicks and seal
them back up by repairing

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the sugar phosphate
backbone of DNA.

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And again, ancient molecular
biologists would prove their

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mettle by purifying ligase and
using it in their reactions.

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And today, where do
we get ligase?

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It's in the catalog.

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Ligase is in the catalog,
reasonably cheap.

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So, when we add ligase we could
now get a hybrid piece

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of DNA that was half human
and half zebra.

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I got to say, this freaks
some people out.

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This doesn't freak me out
because it's just a sequence

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of nucleotides.

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If I give you a sequence of
nucleotides, to my mind,

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there's nothing human
or zebra about it.

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It's a chemical.

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And you can make bonds between
chemicals and all that.

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So, we now pasted our
DNA using ligase.

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Ligase from the catalog.

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Now the question is, what are we
going to paste our DNA to?

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What do we paste our DNA to?

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We're going to paste our DNA to
a fascinating other piece

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of DNA like this.

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I'll make that a little
bigger here.

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That has an EcoR1 site,
has an EcoR1 site.

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And we're going to take our
human DNA and paste it in such

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a way that it makes a circle.

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This piece of DNA is a vector.

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Vector means it travels
around.

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It carries things with it.

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So I want this vector to be
able to replicate if I

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transfer it to E Coli.

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So that's the trick.

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I would like to be able to
attachment my human DNA to

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this vector DNA, and when I
transfer it to E coli, have

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this thing be able to grow.

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So that means we need to invent
a piece of DNA, a

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vector, that is capable
of causing E. coli

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to replicate itself.

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It's got to have all the
instructions to cause E. coli

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to replicate this
piece of DNA.

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So this is another amazing
bit of engineering.

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How do we invent just the right
sequence of letters that

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would allow E. coli to
replicate this thing?

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AUDIENCE: Already
been invented.

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PROFESSOR: Sorry.

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AUDIENCE: It's already been
invented by E. coli.

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PROFESSOR: It's already
been invented by E.

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coli, hasn't it?

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So you're getting the theme
here, is that we don't

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actually do anything in
molecular biology.

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Now look, in fairness, life's
had 3.5 billion years.

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We've been at this a decade
or two, on the

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whole, maybe three.

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On the whole, life's had
a lot more time to

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work this stuff out.

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And usually the best solution
is to look in nature to see

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where nature has already
done it for you.

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So E. coli, it turns out,
replicates its own chromosome.

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You could use the machinery
from its own chromosome.

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But it turns out even
better than that.

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E. coli has the following.

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Here's its big chromosome, four
million letters of E.

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coli chromosome.

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E. Coli also has within
it little circles

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of DNA called plasmids.

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These little circles
of DNA are able to

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autonomously replicate.

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They can copy themselves.

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Or that is to say, E.
coli copies them.

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These little plasmids have the
full replication instructions

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encoded in them.

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Now why does E. coli have
these plasmids?

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What's in these plasmids?

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What's going on?

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Well let's blow up one
of these plasmids.

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It's a big circle.

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And it has an instruction here,
a sequence, called an

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origin of replication - or
amongst friends, just ORI.

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It's called ORI.

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You'll find on maps, origin
of replication.

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That sequence alone is enough to
cause E. coli to open it up

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and start doing its DNA
replication from the origin.

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But what tells you why this is
so important is that these

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plasmids typically have
one or two genes.

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And the one or two genes that
they typically have are genes

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that encode a protein that gives
them resistance to an

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antibiotic, like say,
penicillin.

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There could be a gene that
gives you penicillin

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resistance.

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That's kind of cool.

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E. coli, or other bacteria--

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not E. coli necessarily
for penicillin--

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carry around often
little circles.

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And these little circles encode
genes that give them

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resistance to streptomycin,
penicillin, ampicillin, all

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sorts of things.

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This allows it to grow, even if
you're taking penicillin.

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Now, that's pretty clever.

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How did E. Coli come up?

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How is it so smart to know to
have come up with a gene able

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to break down penicillin, given
that we've only been

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using penicillin since
the 1940s?

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Pretty fast for E. coli to have
figured out how to do

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that, isn't it?

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Oh yeah, who invented
penicillin?

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Nature invented penicillin.

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It's produced by fungi.

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Fungi have been fighting E.
Coli with penicillin for

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millions of years.

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E. coli didn't invent
it for us.

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E. Coli invented it because it's
in a war down there at

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the single cell level
with fungi.

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Fungi make antibi--

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You see, we think
we're so cool.

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We make antibiotics.

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Well we've been making
antibiotics

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only since the 1940s.

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Nature's been making antibiotics
forever.

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Just like the viruses are
infecting E. coli and E. coli

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needs an immune system
against the viruses.

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Well the fungi are throwing
antibiotics at the bacteria

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and it needs a defense
mechanism.

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And the defense mechanism are
these genes that can break

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down those antibiotics.

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And as usual, we just
come along and we

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say, oh look at that.

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It's already been invented.

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Kind of cool.

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We'll use that one too.

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Now why are these on these
little circles?

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They're on the little circles
because when this bacteria has

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lived a long and happy life and
it dies and it spews its

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guts out, neighboring bacteria
suck up the DNA.

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Sounds a little cannibalistic
or something.

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But that's how it
is down there at

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the single cell level.

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The neighboring bacteria
will suck up DNA.

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And why is that cool?

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It's cool because they can
acquire these resistances.

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That's pretty impressive.

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They can acquire
the resistance.

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And in fact, it even works
across species.

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That's why you don't put it
on the chromosome here.

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Because E. coli could pick it up
from another species that's

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not E. coli, but related
enough that it

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can use that plasmid.

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And you can transfer antibiotic
resistance across

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many species of bacteria.

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So the bacteria like to suck up
DNA and see what they find.

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Pretty cool.

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By the way, this is also why
indiscriminate use of

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antibiotics, for example, in
animal feed, or when you were

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given antibiotics by the MIT
health service and are told to

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take them for two weeks and you
take them for five days,

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you're not doing anything
very good.

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You're just selecting for
bacteria that can grow in the

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presence of antibiotics and
selecting for multidrug

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resistance in the spread
of antibiotics.

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We have to be pretty careful.

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Because all these mechanisms
of swapping antibiotic

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resistance are pretty
impressive tricks.

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Anyway, so these things exist.

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And originally, molecular
biologists

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discovered these plasmids.

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They purified these plasmids.

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And the deal was this,
they cut the

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plasmid with EcoR1, ideally.

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So I'm going to put this
up here, vectors.

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They cut the plasmid
with EcoR1.

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They ligate human DNA into it.

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And they get these circles.

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And there you go.

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Now today, obviously, if I
wanted to do this experiment,

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would I purify the
plasmid myself?

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No.

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Where would I get the
plasmid from?

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AUDIENCE: Catalog.

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PROFESSOR: It's in
the catalog.

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There's a whole section
a plasmids here.

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They have vectors
of all sorts.

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So you get it from
the catalog.

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All right.