Hi. It's Mr. Andersen and welcome to part 2 of the new AP Biology labs. We're
going to jump right in with lab 8. In lab 8 what you're doing is bacterial transformation.
Transformation remember is a process by which bacteria can take in DNA from their environment.
Usually they do that by taking in plasmids which are little circles of DNA. And this
is what a plasmid looks like. The one that we'll use in this lab is one that we purchase.
So this was created. It's called pGLO. And it's got four things that are important. This
is where the DNA will start being copied. Right here. This is a resistant to ampicillin.
So if they pick up this plasmid, ampicillin won't kill them. That's going to be an antibiotic.
Next thing they have is an arabinose operon. So that's going to be a part that really is
almost like an on switch. On-off switch. I'll talk about that in a second. And then they
have a glowing fluorescent protein. This is a gene that they took out of a jelly fish
that causes them to glow green. And so if you do this right, your bacteria are going
to glow green under an ultraviolet light. It'll really pop out. And so let's talk about
the engineering and how they figured this thing out. This right here is an operon. If
you're not sure what an operon is, you may want to watch my video on the operon. But
basically what you have is DNA. And this is the promoter region. This is where the RNA
polymerase gets on. This is the operator. And this a series of three genes that they
use. Bacteria just use so they can utilize arabinose which is a sugar. And so what the
scientists did is they took those three genes out and inserted the gene for the GFP, this
glowing fluorescent protein. And so what you can do is if you add arabinose, it will bind
to the repressor. That gets pulled off and then it's going to make the glowing proteins.
And so let's look at how this experiment is set up. What you'll do is you'll take two
petri dishes where you're going to grow normal e. coli. In other words, e. coli that haven't
picked up the plasmid. And then the other two we're going to use transformed. Ones that
we allow them to pick up this plasmid. In the first one we're giving them luria broth.
Luria broth is like a food for bacteria. So you can see that that's found in all four
of these petri dishes. But in this one we're also putting ampicillin. So what do you think
this is going to look like when we try to grow bacteria in it? None of them are going
to be able to grow because the ampicillin is going to kill all of them. What do you
think it's going to look like here when they just get food but no antibiotic? It's going
to form what's called a bacterial lawn. So there's going to be bacteria over the whole
thing. It's simply going to cover it. Let's go to the next one. On this one these are
transformed bacteria. Remember they picked up the plasmid. And so these ones are going
to get the luria broth and the ampicillin and what you'll find is the whole thing isn't
covered, but a lot of it is covered. And these are going to be colonies that come form bacteria
that are now resistant to ampicillin. Because they picked up the plasmid with the ampicillin
resistance. And then this last one is going to be exactly the same except we're giving
them that sugar. And what does that look like? You're going to have bacteria that are actually
transformed and glowing. Because not only are they resistant to the antibiotic, but
we're giving them the sugar so they can turn on the GFP. Next one is going to be restriction
analysis of DNA. This is gel electrophoresis. And so what you do is you take DNA. So a stretch
of DNA and then you cut it with restriction enzymes. Remember restriction enzymes are
used in bacteria in their endless fight against viruses. And so it can cut out certain sections
of the DNA. What's neat about all life is that we all have DNA that can be cut with
the same restriction enzymes. And so in gel electrophoresis what you do is you load up
the DNA on this side of the gel, which really is like jello. You turn on the voltage and
the DNA is going to be attracted towards this positive end. So the DNA is going to start
to migrate through the gel. Eventually you turn it off. And then you're going to have
what is a picture like this. And so what do we have here? This is where they started.
So they started on this side. And then as they move in this direction the smaller fragments
are going to go a farther distance because they're smaller and they can kind of sneak
through this gel. Ones that are bigger are not going to move as far. And so what is cool
is we can put a marker, this is DNA with known distances between the nucleotides or how long
those fragments are. And we can compare it to the DNA that we have in our experiment.
What's a great application of this? Well DNA fingerprinting. And so let's say we have these
three people right here. What is their DNA going to look like? Well we're all humans.
So we're going to have the same genes. But sometimes in between genes we're going to
have restriction fragments. And so these are called restriction fragment length polymorphisms.
So that's a big word. But what it means is sections of DNA that are just repeated. And
we're going to have different numbers of repeats in each human. So we're going to have more
here, less here. More here, less here. More here. So what you do is cut these with a restriction
enzyme and then you run them through a gel. And so let's say a murder has been committed.
And what we do is we find some blood on a murder weapon. We can then do a DNA fingerprint
of that. And then we could do our three suspects over here and we look at where it matches
us. And wherever it matches up horizontally then you've found . . . Okay. Let's going
to the next one. This is 10. Number 10 is on energy dynamics. Energy dynamics is how
numbers, biomass, energy essentially move in ecosystems. And so there's this idea of
a pyramid. So that's a pyramid of energy. Pyramid of numbers. Pyramid of biomass. In
other words at the producer level that's going to be the plants. We're going to have more
biomass. As we move up to the consumers we're going to have less and then we're going to
have less up here. What's an example of that? This could be plants in a grassland. This
could be insects eating that. This could be mice eating those. And this could be a hawk
eating that. And so what we're finding is the energy is dropping off as we go up higher
levels in the food web. Why is that? We're losing energy at each of those levels for
life. And we're losing it as heat. Likewise up here we're losing a lot of that mass just
through respiration to carbon dioxide. And so in this experiment, what you're going to
look at first is primary productivity. So that's going to be productivity of the producers.
And a good way to measure that indirectly is to measure the amount of oxygen they're
producing. Because if we measure the amount of oxygen they are producing we can just kind
of figure out using some quick math how much carbon they're actually producing. And so
in this experiment we're going to have two beakers. Each of these have, or two flasks.
Each of these have algae in it. And they start with a dissolved oxygen level of 6.0 milligrams
O2 per liter. What we'll do next is we'll cover up one of these. And this is going to
be the dark beaker. And then we're going to apply sun to it. And watch what will happen
right in here. The oxygen levels are going to go up. And so what we measure is the primary
productivity. In other words primary means at that that first level, the amount of energy
that they're producing. And so in this case the net primary productivity is going to be
to 2 milligrams of oxygen per liter. How do I figure that out? It's the amount after the
experiment minus the amount that it had to begin with. It's ow much it's gained. So what
is gross? That's another one. Gross primary productivity is going to be the total. And
so an analogy might work here. Let's say you have a job and you make $100,000 in a year.
That's going to be the gross pay that you make. But things are going to eat in on that.
So you've got taxes, social security, a car payment. And so you're only going to net a
certain amount of money. And so same thing occurs in the beaker. So this one gained 2
milligrams oxygen. It looks like that's our net. But is we subtract 8 minus 5, that's
going to give us our gross. Well where's that coming from? Well remember these algae are
doing photosynthesis to create more oxygen. But they're also doing respiration. And that
respiration is going to consume it. And so the bottle that was sitting in the dark is
losing oxygen just to respiration. And so we can figure out that gross by subtracting
this light beaker after the experiment minus the dark beaker. Next one on that is looking
at the biomass change. And I didn't do this is my lab, but this is kind of how it works.
What you do is you take some brussels sprouts and you're going to apply a caterpillar to
these. So you're going to get the mass of the brussels sprouts and the caterpillars,
these are like a white cabbage butterfly. And what they'll do is that they'll eat on
the brussels sprouts and you can get a mass of the caterpillars, how much mass is there?
And then a mass change in the plants and we can figure out that next level as far as biomass.
That you're losing it on each level. And they're losing that to respiration and processes.
Next one is transpiration. In the transpiration lab what you're measuring is transpiration
using a potometer. And so this is a leaf connected to a tube of water. And as water evaporates
off the top of the leaf that water is going to be consumed and so we're going to see this
side go down. So we can measure the rate at which water is evaporating off a leaf. Now
how does it evaporate off the leaf? Through these things which are called stomata. Stomata
are going to be little openings in the leaf. And as they open up you can let more water
evaporate off. That's going to be a higher rate of transpiration. As they close it's
going to eventually stop. And so in this lab what we're looking at are a control. So this
is going to be just room normal. We're then going to have one where we put a bag around
the outside and increase the humidity inside it. And what's going to happen there is it's
going to slow down the rate of transpiration. Just because what we're doing is we're increasing
the water potential. So there's not much of a draw towards that evaporation. In the other
two we're going to add light. And that's going to speed up transpiration. And then a fan
or wind, which is going to speed it up. So you may want to think of anything that increases
evaporation is going to increase rate of transpiration. Here's a quick graph. And what you can do
is calculate again the slope. And that's going to tell you the rate of the reaction. They
next one we do is animal behavior. The old school way to do this is using these pill
bugs. What you do is you put pill bugs on either side of the what's called a choice
chamber. You put five on each side. And you allow them to move through the middle. What
you then put is a treatment. So we can put wet on this side and dry on this side. And
we just let them go. Every half a minute we're going to record how many you have on each
side of the choice chamber. Now pill bugs show what's called kinesis. In other words
they randomly move around when they're unhappy. And when they're happy they stop. And so this
is some data that I collected. On average you're going to have 9 bill bugs on the wet
side. And on the dry side you're only going to have one. So you could do Chi-Squared analysis
to figure out are they really choosing the wet side over the dry side. What is taxis?
Taxis is when you're going towards a specific stimuli. And so in this experiment you could
have a choice chamber as well with fruit flies. You introduce the fruit flies in the middle
and then you put different smells on either side. And so these chemicals could be alcohol,
vinegar, water. You could put ripe fruit. And so what you do is you try different things
on either side and you record the number of fruit flies that go to each side. We can put
that along what's called a matrix. And so we'd have the substance on side A, the substance
on side B and we can figure out which ones are they choosing. And so A, if we say that
is water, we might have 5 and 5. 5 on each side. And that just kind of acts as our control.
But we can figure out which substance do they prefer by looking at and comparing it to substance
A and B. So let's say this is 7 and this is ripe fruit. So we've got 10 in the ripe fruit
side. 0 on the control side. And so we can eventually say that they're choosing ripe
fruit. Next one and the last one is the enzyme activity. So in this one what we're using
is an enzyme. You could use catalase. That's what we use. We extract it from yeast. Or
you could use peroxidase. These essentially do the same thing. They take hydrogen peroxide
and break it down into water and oxygen. Peroxidase does that kind of indirectly using an organic
carrier. Catalase just does it directly. And what you're going to do is your going to measure
the rate of the enzyme action. And so how I do that is we dip our little chads, these
are paper chads now, in different concentrations of the enzyme. Different concentrations of
this yeast solution. We then put them in a beaker that contains hydrogen peroxide. They
break that hydrogen peroxide down into water and oxygen. The oxygen bubbles build up on
the chad and eventually it floats. And so we could measure the rate of respiration or
excuse me, the rate of enzyme action. So down here we could put concentration. Up here we
could measure how long it takes for those chads to float. I measure the units here in
floats per second. And we could figure out the rate of this reaction. What we'll find
is as you increase this, it's eventually going to level off. Where if you get so much enzymes
it doesn't really matter. There's too many of the enzymes for substrates. So other things
you could vary or have varied in here is you could look at concentration. You could look
at temperature. You could look at pH and see how that's going to effect enzyme action.
And so those are the labs. I hope that was helpful.