WEBVTT

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PROFESSOR: Good morning,
good morning.

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So last time, we talked about
the most remarkable

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biochemical purification
procedure ever invented--

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cloning.

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You remember the issue
with biochemistry.

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You're going to grind up a cell,
you're going to take the

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contents and run it over
different kinds of separation

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columns or centrifuge it or
things, in order to separate

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some things from other things.

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The problem with purifying a
gene that way, away from all

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the rest of DNA in a cell, is
that the gene has exactly the

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same biochemical properties
as all the other genes.

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How in the world are
you going to do it?

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The solution was devilishly
simple and devilishly complex

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at the same time.

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All you have to do to purify
something is dilute it.

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If I take any substance
an I add enough

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water, it's very dilute.

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And in any little drop in any
little test tube there will

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only be one DNA molecule.

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It's now purified.

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I could do that for every
molecule there is.

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The problem is, it's not very
useful unless I have a way to

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take that molecule and make
extra copies of it.

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But if I could do that, dilution
is purification.

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The trick we use initially for
making more copies is we ask

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E. coli to do it for us.

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That, as usual, is the solution
to most issues.

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Find something in life
that already does it.

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And so, just to remember what
we did, we took total DNA.

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Maybe it was yeast DNA, maybe
it was human DNA, maybe

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it was zebra DNA.

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And we cut it up with some
restriction enzyme.

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Our restriction enzyme we used
was EcoR1 And it therefore cut

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at the EcoR1 sites, G, A, A, T,
T, C. We could have used a

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different restriction enzyme.

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We then added it to a vector.

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The vector was cut open
at an EcoR1 site.

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The vector had an origin of
replication, ORI, and the

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vector had a resistance marker,
some resistance gene

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that made a protein that could
break down some antibiotic

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found in nature.

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We combine these two pieces.

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Vector now gets its insert.

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We call this vector, we
call this insert.

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We attach them together
using what?

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AUDIENCE: Ligase.

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PROFESSOR: Ligase, the enzyme
that ligates DNA.

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We then take this, we transform
it into a bacterium.

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The scale is obviously
off, right?

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This DNA plasmid here is tiny
compared to this bacteria.

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But if I draw it to scale,
it won't be very helpful.

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So we then transform
it in here.

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We do that in a test tube.

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We treat the bacteria a little
roughly so it likes to slurp

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up the DNA.

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We then plate it on a plate.

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And those bacteria that have
acquired our plasmid

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containing the antibiotic
resistance gene are able to

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grow on this plate that
has antibiotic on it.

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And we call this thing
a library.

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So that's it.

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We can make a library.

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And we have in effect,
then, what I said.

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We've deluded the individual
molecules out, and each one

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went into its own bacteria, and
each one got replicated by

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that bacteria.

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Those plasmids are replicated
by the bacteria.

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In fact, we choose plasmids
that are called multicopy

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plasmids, where there's not
just one copy but the cell

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might make 50 copies of it.

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We grow up a whole colony
of it and there you go.

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We talked about some of
the issues with it.

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Where do we get the restriction
enzymes, the

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ligases, the vectors,
et cetera?

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It's in the catalog, right?

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We used to purify them
ourselves, but they're all in

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the catalog.

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So any questions about this?

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We had some questions.

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I have a question about this.

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How come when we add ligase the
vector itself just doesn't

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close up into a circle without
an insert in it?

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It might.

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What would happen then?

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AUDIENCE: You'd just have
your original vector.

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PROFESSOR: You'd just have
your original vector.

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And what happens when I
transform into the bacteria?

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AUDIENCE: It'll still survive.

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PROFESSOR: It'll
still survive.

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So will the unimolecular closure
of that circle be more

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common than the bimolecular
interaction between a vector

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and an insert?

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AUDIENCE: Probably.

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PROFESSOR: Probably, because the
two ends of the circle are

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pretty close to each other.

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So what do I do?

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Yeah?

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AUDIENCE: Because [INAUDIBLE]

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are the same.

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[INAUDIBLE]

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same restriction enzymes
because if they're two

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different enzymes so
they don't match.

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PROFESSOR: Ooh.

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That's cute trick.

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Two different restriction
enzymes so that

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they couldn't reclose.

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But then my fragments better
have those two different

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sites, too, and only be able to
clone those fragments that

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have the two different ones.

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But that could work.

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So you want a trick for making
sure it doesn't reclose.

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Any other tricks?

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But the problem is, I won't get
all the fragments, only

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the ones that have, say,
an Eco at a Bam site.

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But that's--

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So I bring this up not because
it's particularly important,

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but to tell you the kind of
engineering that really does

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have to go on in molecular
biology.

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What happens is when you have
your DNA put in here, we have

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a sugar-phosphate backbone
in both cases.

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And if we look up close, one
of these sides has the

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phosphate, the other
has a hydroxyl.

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Phosphate, hydroxyl, phosphate,
hydroxyl, right?

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And ligase comes in
and joins that.

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So this guy has a phosphate on
this strand, and this guy has

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a phosphate on that
strand, hydroxyl

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there, hydroxyl there.

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What would happen if
I got rid of the

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phosphates on the vector?

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Could it reclose?

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AUDIENCE: No.

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PROFESSOR: No.

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So if there were an enzyme that
removed phosphates, I

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could treat my vector
first with the

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phosphate-removing enzyme.

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And now it couldn't possibly
reclose on itself.

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And is there such an enzyme?

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And what's it called?

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Phosphatase, because it takes
phosphates so easy.

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Phosphatase, it takes
off the phosphates.

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And then it can't
reclose anymore.

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And where do we get
phosphatase?

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It's in the catalog.

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Exactly.

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So now what happens
is that the vector

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only has an OH here.

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What happens to ligase?

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When I put an insert in here,
ligase can make a covalent

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join on this strand.

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But it can't actually
make a covalent join

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on the other strand.

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But does it matter?

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No, because I've closed up my
circle on that strand, and I

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close up the circle there on the
other strand, and I just

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throw it into E. coli.

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And you know what happens when
it goes into E. coli?

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It's got a nick, obviously,
on that strand.

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It hasn't closed up.

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But what does E. coli do
when it sees that DNA?

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Must be damaged DNA.

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I'll fix it.

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So E. coli actually does the
last little trick of closing

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that up for you with
its own enzymes for

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repairing its own DNA.

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I bring this up not because it's
crucial that you should

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worry about it, but because I
want to know that there's a

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whole layer of these interesting
engineering tricks

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that get developed.

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Every one of them exploits
enzymes that we know.

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Every one of them deals with
questions like, will my vector

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reclose on itself, how
do I avoid that?

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And there's a vast cooking
book of protocols

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in molecular biology.

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And we constantly are just
cribbing from things life does

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to make our protocols more
and more efficient.

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So I bring it up more because
it's kind of a cool thing that

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all that goes on, and because
it helps you remember that

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these phosphates are
very important to

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joining things up.

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That's a digression.

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Now, let's go to the topic.

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How do we actually
read the library?

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How do we read our library?

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How do we use the library,
read from the library?

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Well let's say we're going to
try to find the arginine gene.

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We talked about the gene
for arginine in yeast.

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So I'd like to clone
the gene for ARG1.

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We found mutants before that
were unable to grow without

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supplemental arginine.

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They somehow had a defect in
producing their own origin.

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It's a mutant.

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I want to find the
gene, please.

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How do I do it?

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We've got to think about
what's our insert DNA.

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What are our vectors?

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What insert DNA should
we start with, zebra?

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No.

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Human?

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No.

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How about yeast?

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We're trying to clone a gene
from yeast, right?

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So let's start with yeast.

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OK.

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So we're going to start
with yeast DNA.

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We're going to cut
up yeast DNA.

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We're going to attach it to
our vector, we're going to

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transform it into E.
coli, E. coli will

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grow up on our plates.

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And one of these guys, I happen
to know it's that one

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there, contains the ARG1 gene.

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The problem is I happen to
know it, but you don't.

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How are you going to find out
where the ARG1 gene is?

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Any takers?

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Yeah?

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AUDIENCE: It could be like when
you put the gene in make

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it flourescent.

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PROFESSOR: A fluorescent tag?

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So I should just attach
the fluorescent

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tag to the ARG1 gene?

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AUDIENCE: Yes.

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PROFESSOR: How do I do that?

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All the DNA looks the
same in the tube.

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How do I know where to attach
the fluorescent tag?

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AUDIENCE: Maybe you could
size it [INAUDIBLE].

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PROFESSOR: There's a lot
of pieces of DNA there.

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And my eyes are not that good.

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AUDIENCE: Separate it?

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PROFESSOR: Separate it.

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But will I know which
one is ARG1?

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See, I don't actually know
anything about ARG1.

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All I know is I made a mutant.

00:11:40.870 --> 00:11:43.370
The mutant is unable to
grow without arginine.

00:11:43.370 --> 00:11:45.800
I haven't got a clue
what that gene is.

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I don't know what it encodes, I
don't know how big it is, I

00:11:48.040 --> 00:11:49.550
don't know nothing.

00:11:49.550 --> 00:11:54.430
All I know is that whatever it
is, it's a gene which when

00:11:54.430 --> 00:11:58.176
mutated prevents you from
growing without arginine.

00:11:58.176 --> 00:12:01.032
AUDIENCE: Could you plate all
of your colonies onto a--

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could you put [INAUDIBLE]?

00:12:03.412 --> 00:12:04.510
PROFESSOR: Minimal medium.

00:12:04.510 --> 00:12:07.740
What if I plate on
minimal medium?

00:12:07.740 --> 00:12:08.740
Now what?

00:12:08.740 --> 00:12:09.665
What are you hoping for?

00:12:09.665 --> 00:12:14.560
AUDIENCE: The one that has the
ARG1 gene will not grow.

00:12:14.560 --> 00:12:17.760
PROFESSOR: The one that
has the ARG1 gene

00:12:17.760 --> 00:12:20.180
won't be able to grow--

00:12:20.180 --> 00:12:21.170
oh wait, yeah.

00:12:21.170 --> 00:12:22.810
But something like that.

00:12:22.810 --> 00:12:23.850
Let's work it through.

00:12:23.850 --> 00:12:24.830
We've got my idea here.

00:12:24.830 --> 00:12:26.992
What are we going
to do with it?

00:12:26.992 --> 00:12:28.242
AUDIENCE: [INAUDIBLE]

00:12:32.460 --> 00:12:36.830
PROFESSOR: I've got a working
ARG1 Mutate ARG1 afterwards?

00:12:36.830 --> 00:12:39.490
AUDIENCE: [INAUDIBLE]

00:12:39.490 --> 00:12:40.200
PROFESSOR: OK.

00:12:40.200 --> 00:12:41.350
How will that work?

00:12:41.350 --> 00:12:43.170
I'm open for--

00:12:43.170 --> 00:12:44.340
got an idea here?

00:12:44.340 --> 00:12:46.116
AUDIENCE: If you have your
different colonies

00:12:46.116 --> 00:12:50.456
[INAUDIBLE], you could have a
secondary plate them all over

00:12:50.456 --> 00:12:51.440
to one of middle medium.

00:12:51.440 --> 00:12:56.160
The ones that die are the ones
that already [INAUDIBLE]--

00:12:56.160 --> 00:12:58.850
PROFESSOR: So guys,
I have a concern.

00:12:58.850 --> 00:13:02.190
I'm just transferring
this into E. coli.

00:13:02.190 --> 00:13:04.490
E.coli grows just fine
without arginine.

00:13:07.630 --> 00:13:09.800
I mean, I'm going to take
this yeast DNA.

00:13:09.800 --> 00:13:10.880
I'm going to put
it in E. coli.

00:13:10.880 --> 00:13:13.290
E. coli was kind enough
to grow it for me.

00:13:13.290 --> 00:13:17.800
But frankly, E. coli doesn't
need this ARG1 gene.

00:13:17.800 --> 00:13:19.200
E. coli can grow without
arginine.

00:13:19.200 --> 00:13:21.520
I can plate this with and
without arginine, E. coli

00:13:21.520 --> 00:13:22.930
grows just fine.

00:13:22.930 --> 00:13:24.150
But you're on to something.

00:13:24.150 --> 00:13:27.140
You're onto the idea that
somehow, the only thing we

00:13:27.140 --> 00:13:34.120
know about ARG1 is that the
functional, wild-type copy of

00:13:34.120 --> 00:13:40.570
that gene confers an ability
to grow without arginine.

00:13:40.570 --> 00:13:44.110
And who does it confer it on?

00:13:44.110 --> 00:13:46.820
And what kind of yeast?

00:13:46.820 --> 00:13:51.420
Haploid mutant yeast.

00:13:51.420 --> 00:13:52.570
Ah.

00:13:52.570 --> 00:13:57.310
So suppose I put a working copy
of ARG1, a good copy, a

00:13:57.310 --> 00:14:01.770
wild-type copy, into
a mutant yeast.

00:14:01.770 --> 00:14:04.080
Now what would happen to
that mutant yeast?

00:14:08.270 --> 00:14:08.780
What would happen?

00:14:08.780 --> 00:14:12.260
The mutant yeast before, could
it grow without arginine?

00:14:12.260 --> 00:14:13.100
No.

00:14:13.100 --> 00:14:14.990
If I put in a working
copy of the ARG1

00:14:14.990 --> 00:14:17.650
gene, what will happen?

00:14:17.650 --> 00:14:19.320
It grows.

00:14:19.320 --> 00:14:20.720
Now let's design a scheme.

00:14:25.820 --> 00:14:28.040
Do I want to use
E. coli at all?

00:14:30.570 --> 00:14:31.870
No.

00:14:31.870 --> 00:14:34.226
What do I want to use?

00:14:34.226 --> 00:14:35.670
I want to use a yeast.

00:14:35.670 --> 00:14:40.330
So let's get rid of E. coli and
let's instead use yeast.

00:14:40.330 --> 00:14:44.140
And which yeast should we use,
wild-type or mutant?

00:14:44.140 --> 00:14:45.280
Mutant yeast.

00:14:45.280 --> 00:14:47.280
What mutation?

00:14:47.280 --> 00:14:50.210
ARG1 mutant yeast.

00:14:50.210 --> 00:14:53.000
ARG1 minus yeast.

00:14:53.000 --> 00:14:57.740
Now, if I plate ARG1 minus
yeast on minimal

00:14:57.740 --> 00:15:00.330
medium, what happens?

00:15:00.330 --> 00:15:01.830
It doesn't grow.

00:15:01.830 --> 00:15:04.520
It dies.

00:15:04.520 --> 00:15:08.585
What DNA should I
be putting in?

00:15:08.585 --> 00:15:11.070
Yeast DNA.

00:15:11.070 --> 00:15:13.370
Mutant or wild type?

00:15:13.370 --> 00:15:16.110
Why wild type?

00:15:16.110 --> 00:15:19.070
Because it'll have a working
copy of ARG1.

00:15:19.070 --> 00:15:22.600
So I want yeast, wild type.

00:15:22.600 --> 00:15:24.410
Now what happens?

00:15:24.410 --> 00:15:27.680
One of these guys, and only one
of these guys here, this

00:15:27.680 --> 00:15:31.970
one, has a ARG1 gene.

00:15:31.970 --> 00:15:33.840
That's ARG1.

00:15:33.840 --> 00:15:40.720
When it goes in, that plasmid
has the ARG1 plus gene,

00:15:40.720 --> 00:15:44.430
whereas other plasmids don't.

00:15:44.430 --> 00:15:47.550
That cell that inherits that
gene there, that gets that

00:15:47.550 --> 00:15:49.320
gene, is not green.

00:15:49.320 --> 00:15:51.050
I just drew it green for you.

00:15:51.050 --> 00:15:52.850
But it has the ARG1 gene.

00:15:52.850 --> 00:15:54.950
And when I plate this on
minimal medium, what's

00:15:54.950 --> 00:15:56.650
distinctive about it?

00:15:56.650 --> 00:15:58.280
It grows.

00:15:58.280 --> 00:16:01.470
That's how you can clone
the ARG1 gene.

00:16:01.470 --> 00:16:03.910
You clone it by the only thing
you know about it.

00:16:03.910 --> 00:16:06.520
Namely, it confers a function.

00:16:06.520 --> 00:16:10.880
This is called cloning
by function.

00:16:10.880 --> 00:16:22.290
Or, what did we do when we
crossed two mutants together

00:16:22.290 --> 00:16:26.500
to see if things were
in different genes?

00:16:26.500 --> 00:16:29.990
It was a test of
complementation.

00:16:29.990 --> 00:16:34.040
Really what we're asking is,
is there a plasmid that

00:16:34.040 --> 00:16:38.400
complements the defect?

00:16:38.400 --> 00:16:44.180
In effect what's happening is
in this cell, right here, we

00:16:44.180 --> 00:16:45.760
have a yeast cell.

00:16:45.760 --> 00:16:50.470
And the yeast cell has
a defect in its ARG1.

00:16:50.470 --> 00:16:55.760
But the plasmid has
a working ARG1.

00:16:55.760 --> 00:17:00.140
So for that one gene, this cell
could be thought of maybe

00:17:00.140 --> 00:17:03.700
a little bit like a diploid,
just at that one gene.

00:17:03.700 --> 00:17:07.569
And we've done a
complementation, just a teeny

00:17:07.569 --> 00:17:09.780
little complementation
for one gene.

00:17:09.780 --> 00:17:13.920
And we could call this cloning
complementation.

00:17:19.930 --> 00:17:21.405
It's essentially cloning
by function.

00:17:30.380 --> 00:17:32.190
Any questions?

00:17:32.190 --> 00:17:33.884
Yes?

00:17:33.884 --> 00:17:36.369
AUDIENCE: [INAUDIBLE]

00:17:36.369 --> 00:17:40.345
--they all have functioning
[INAUDIBLE]?

00:17:40.345 --> 00:17:41.836
PROFESSOR: Yep.

00:17:41.836 --> 00:17:44.840
AUDIENCE: So why does that
then have a function?

00:17:44.840 --> 00:17:45.520
PROFESSOR: Oh, oh.

00:17:45.520 --> 00:17:48.800
You see, the yeast genome has
about 4,000 different genes.

00:17:48.800 --> 00:17:50.910
I chop it up with my EcoR1.

00:17:50.910 --> 00:17:56.090
Some plasmids get ARG1 but most
of them get leucine 2 or

00:17:56.090 --> 00:17:57.200
[INAUDIBLE]

00:17:57.200 --> 00:17:58.460
or other things.

00:17:58.460 --> 00:18:02.040
And each yeast cell in my
library only picks up a

00:18:02.040 --> 00:18:05.160
plasmid with one chunk
of DNA, one gene.

00:18:05.160 --> 00:18:09.180
So it turns out that the yeast
cells in my library, each one

00:18:09.180 --> 00:18:12.080
has one of thousands of
alternative possibilities.

00:18:12.080 --> 00:18:14.480
And it's just the guy who
gets ARG1 who grows.

00:18:14.480 --> 00:18:17.360
AUDIENCE: But you're saying
that the yeast [INAUDIBLE]

00:18:17.360 --> 00:18:18.320
plasmid.

00:18:18.320 --> 00:18:21.210
All code for--

00:18:21.210 --> 00:18:21.780
PROFESSOR: That's right.

00:18:21.780 --> 00:18:24.570
AUDIENCE: But I don't get why
you would end up with

00:18:24.570 --> 00:18:26.500
[INAUDIBLE].

00:18:26.500 --> 00:18:27.250
PROFESSOR: What do I--?

00:18:27.250 --> 00:18:29.162
AUDIENCE: Why do you end
up with a strain

00:18:29.162 --> 00:18:30.600
that has ARG1 in it?

00:18:30.600 --> 00:18:31.090
PROFESSOR: Oh.

00:18:31.090 --> 00:18:33.830
ARG1 is the ARG1 working copy.

00:18:33.830 --> 00:18:37.350
In the yeast, I'm talking about
this yeast here has the

00:18:37.350 --> 00:18:41.160
working copy of ARG1,
ARG1 plus.

00:18:41.160 --> 00:18:44.200
So this guy has an ARG1 plus.

00:18:44.200 --> 00:18:46.610
It also has lots
of other genes.

00:18:46.610 --> 00:18:49.330
Each of these plasmids
gets one gene.

00:18:49.330 --> 00:18:52.150
Some of them get an ARG plus.

00:18:52.150 --> 00:18:55.500
Some of them happen to get a
leucine gene or some other

00:18:55.500 --> 00:18:56.690
gene that's irrelevant.

00:18:56.690 --> 00:19:01.544
And the plasmids that contain
the working copy of the gene,

00:19:01.544 --> 00:19:04.600
they, when they go into the
cell, give the cell the

00:19:04.600 --> 00:19:05.930
ability to grow.

00:19:05.930 --> 00:19:07.005
So that's why.

00:19:07.005 --> 00:19:07.710
All right.

00:19:07.710 --> 00:19:09.980
So that's how we get
a gene by function.