Hi. It's Mr. Andersen and welcome
to AP Biology Lab 6 walkthrough. This is on
molecular biology. So it's the study of this
material, deoxyribonucleic acid. That genetic
material that makes life possible. In this
lab there are actually two parts to it. There's
the transformation part where we can transform
a bacteria. Make it different using a plasmid.
And then gel electrophoresis. How we can separate
DNA. How we can digest it. Break it into fragments
and then separate it according to the length
of those fragments. And so let's get started.
The two things we'll do will be bacterial
transformation. Transformation is when you're
taking one bacteria, so let's draw an e. coli
that looks like this. And here's another e.
coli. And we're transforming this one by passing
DNA between the two. In this case we'll pass
a little bit of DNA called a plasmid from
one to another. The plasmid we'll be using
is something called pglo. So it's a way to
actually get this bacteria to glow fluorescent
glow under a black light. Second thing we'll
do is gel electrophoresis. Gel electrophoresis
is separating DNA according to its fragment
lengths. So we're basically running DNA through
a gel. It's almost like jello. And then we're
separating according to the length of those
fragments. So those are the two things that
we're doing. The bacterial transformation
we're doing is using a plasmid. This is the
plasmid right here. It's called pglo. Basically
pglo is a section of DNA. So in a bacteria
they're going to have their regular DNA. But
they're also going to have these little bits
called plasmids. Plasmids give them the ability
to do something. So fertility plasmid would
be an example. Or resistant plasmids. So that's
going to be one that gives bacteria antibiotic
resistance. And so in this plasmid it's been
engineered. In other words it's been built
by humans. On one bit of the plasmid we're
going to have the origin of replication. That's
where it's copied. These ones are also going
to have ampicillin resistance. So when we
grown them on ampicillin, only the ones that
have picked up the plasmid are going to grow.
Here we've got the glowing fluorescent protein.
This is extracted from a jelly fish. And then
they also have a trigger, a arabinose sugar.
So if you add arabinose sugar they're going
to make that glowing fluorescent protein.
If there's no arabinose then it's not going
to show up. And so basically what we'll do
is we'll have four different types of bacteria.
The bacteria we're using is e. coli, Escherichia
coli. So basically we're going to take some
of the e. coli and we're not going to give
it the plasmid. So it gets no DNA. We'll plate
some of that on what's called luria broth.
That's just basically food for bacteria. And
then we'll plate some of it on luria broth
with ampicillin. So since it has an antibiotic
it's not going to be able to grow. We'll then
add the plasmid to some of the bacteria. We'll
grow some of them on luria broth and ampicillin.
Some will be able to grow. Those that pick
up the plasmid. And then were also going to
have some of those with this trigger, this
arabinose sugar as well. And I'll show you
what all those things are going to look like
in just a second. But first let's discuss
it. So basically what you get is a plasmid.
A plasmid is a little bit of DNA. We're going
to add it to bacteria. And so the protocol
for doing that basically entails putting them
on ice, the bacteria. You then add a transforming
liquid. It's calcium chloride is what it is.
And that causes them to kind of loosen up
their membrane and more likely take in that
plasmid. We're also going to heat shock them.
So they'll be on ice, then we'll heat shock
them. And some of them will pick it up. And
when I do this in class, you know it's two
or three groups out of 20 or 30 groups will
actually get it to transform. So there's a
lot of chance to that. But if you get one
bacteria to pick it up you can get a colony
of that. And then it's going to grow. And
so basically let's kind of talk through it.
If bacteria doesn't pick up the DNA, what
should it look like here? Well we should have
a lawn of bacteria. If ampicillin we should
have no growth here because unless they get
that ampicillin resistance they're not going
to be able to grow. LB/Ampicillin, the ones
who get the plasmid should be able to grow
if they're transformed. And then these are
going to be the only ones that actually glow.
And so if we plate them on it what we find
is that the ones that actually didn't get
the plasmid, but they're on luria broth, what
you'll get is they'll cover the whole plate.
And you can kind of see that on here. But
once we try to grow on ampicillin, the ampicillin
is going to kill them. In other words it's
going to kill all of them, since none of them
picked it up. Likewise on the ones who have
the LB and the ampicillin, the ones that are
transformed, we're going to see some colonies.
So each of these colonies represent one bacteria
that picked up the plasmid. It's able to make
copies of itself. It's able to grow. So you
can see not a ton of them are able to do that.
But then finally we get the really cool plate
where we got LB/Ampicillin. So all of these
bacteria were transformed. But we also have
that arabinose sugar. So they're able to use
that to produce this glowing protein. And
so you get this fluorescence in the bacteria.
And so what do you need to understand? What
a plasmid is. How it transforms bacteria.
And then how that might be manifested in all
of the different growth on all the different
plates. Second part of the lab is called gel
electrophoresis. Gel electrophoresis is going
to use a gel box that looks like this. There
will actually be liquid in it. And then you'll
have a little fragment of jello. This is an
agarose gel. So this is what it would look
like. You're going to load up the DNA in these
wells on one side. And then they're going
to migrate across. They're going to be pulled,
since they have a negative charge, they're
going to be pulled towards the positive end.
And so we let it run for a certain amount
of time. Now if we were to let just DNA run,
what you'd get is one fragment being pulled
across. The smaller fragments of DNA are going
to be able to make it farther. And so the
smaller it is the farther it's going to migrate.
So we know that this one right here is a smaller
fragment of DNA than this one is. But we also
want to trim the DNA. And so we use something
called a restriction enzyme. Restriction enzymes
come from bacteria. Basically bacteria are
constantly being infected by viruses so they
use restriction enzymes to cut up all of their
DNA, remove the viral DNA, and then glue it
back together again. And so basically a restriction
enzyme is going to cut between the nucleotides.
So it's going to cut the DNA in specific points.
But it's only going to cut the DNA when it
has the specific reading frame right here.
Where it's GAATTC. And you can look that that
reads it in the opposite direction on the
other side. So basically we can digest it
using a restriction enzyme. That's going to
cut the DNA up into a little bit. If we were
to use a different restriction enzyme, it
would cut it up into smaller bits or different
bits. And so if we were to run all of this
through a gel, well this would be one big
fragment. So this would not move very far
through the gel. But this fragment way up
here is very small and so it would be migrating
even farther. And then these ones would be,
I don't know, somewhere in the middle. Something
like that. So this would be how this DNA would
separate. If we were to use a different restriction
enzyme we'd cut it up differently. Now you
just use micro amounts of DNA. So we use one
of these micro pipettes to get tiny amounts
of DNA. You then essentially load it in here.
And it's pulled off to the other side. Let
me kind of quickly talk about analysis. Another
thing that a lot of the time we'll load whenever
we're running a gel is something called a
ladder or a DNA ladder. DNA ladder is going
to be engineered. It's sent to you. And each
of those fragments is going to have a different
length. So this would be a 200 base pair.
300 base pair. So this would probably be 400
base pair. So these are known quantities of
DNA. So this would be the ladder on one side.
And then you run your DNA in another lane.
So we can see that this one right here, we
can kind of interpret between these two that
this is maybe 250 base pairs, or 250 nucleotides
between it. Or you could look over here. Here's
another gel. So we loaded it right here in
the wells. It's moved in this direction towards
the positive end. You can see here's our ladder
on this side. But you can see that we cut
it with a different restriction enzyme. In
this case we're using a plasmid. So that's
just that little section of DNA. We use different
restriction enzymes. So it's the same DNA,
but by using different restriction enzymes
they went a different amount. This one looks
like it broke into two. This is maybe just
one. And this might be, I don't know, two
something like that. And so basically we can
do analysis. We can compare that to the DNA
ladder on the side. And we can figure it out.
That's it. So that's molecular biology. That's
the gel electrophoresis part. And I hope this
all is helpful.
