Introduction To Biology15

Good morning, so I see we have a lot of parents here.
How many parents have we got here? Welcome to the parents.
How many of the parents have done the reading for today?
Good, ‘cause I'll call on the parents, too. Right? 
We'll see what happens.All right, so. Where are we?　
We've talked about this diagram that I keep coming back to.
If you want to study biological function, the two traditional ways to do that.
We'd look at genetics. We'd look biochemistry.
Genetics, the study of an organism with one broken components,
those components being genes.
Biochemistry, the study of the purification of individual components,
 from an organism, away from an organism,
Particularly the most important such components being proteins.
What do they have to do with each other.
The unification in molecular biology that occurred in the middle of the century,
from 1950s' to the 60s', and really up to 1970s'.
And so we came to a conceptual understanding that genes encode proteins
And therefore these two different ways of looking into the organism,
organism minus component, component minus organism,
were complementary points of view.
And in theory, you could go from a gene sequence to a protein sequence,
a protein sequence back to a gene sequence.
You could go from gene sequence to its function,
its function to a protein, except for one tiny detail.
This was all just conceptual.
Conceptually we understood by about 1970 that the DNA made the RNA made the protein.
The protein carried out the function.
But as of then, you couldn't individually work with,
or purify the DNA corresponding to any particular gene.
All the influences had been indirect influences.
indirect influences from bacteria genetics, bacteria regulation or Meselson-Stahl experiments.
All sorts of interesting indirect ways working out the genetic code.
But it didn't let you read anything. This was the problem.
Some people of late 1960s' said: Great molecular biology is over.
We understand the principle how life works.
Now we're going to understand how the brain works.
And there was an exodus of some people from molecular biology into neurobiology,
to now go nail the brain. Figuring that out will be worth another ten years or so ***
But in fact, remarkably, people began to focus on how you could get to work with individual specific genes.
Now what's so hard about that.
I mean it's not very hard to crack open a red blood cell and purify different proteins.
You can purify hemoglobin. You can purify different enzymes.
Biochemistry allows you to purify different components from each other.
I want to purify an enzyme.
Let's crack open the yeast cell, separate the proteins over some columns
that separates them based on their size, or their charge, on their 03:05 fractions
or assay each fraction to see which one has the enzymatic activity.
But these guys use the physical-chemical properties of the proteins
to separate them into different buckets.
Why not do that with, say, the human DNA
and purify out the gene for betaglobin,
that encodes the beta component of hemoglobin?
What would be the problem of just using physical-chemical purification
to purify one human gene from another?
Well this is one very big molecular.
I mean I can shear it up maybe I'll just break it up.
Now let’s purify the betaglobin containing part.
It all looks the same. It's just DNA.
It's one chemical polymer with pretty boring properties.
And they are not very different.
Any particular DNA sequence and any other DNA sequence,
basically about the same molecular weights and charges.
There is nothing to separate them.
How are you gonna purify betaglobin? That was the problem.
That's where recombinant DNA came in
Recombinant DNA was a remarkable and totally different way of purifying individual components.
And the basis of it was this notion of cloning.
If I want to purify out from the human genome,
How big is the human genome?
Human genome's about 3 billion bases long.
If I want to purify a particular gene, let's say betaglobin or some other gene,
typical gene, might be on the order of 30 thousand letters long.
This is one part in 1e5 purification I’ve got to achieve.
Any given gene is only one part of 1e5 of the human genome.
And what about a typical mutation?
Maybe the mutation that causes sickle cell anemia by changing a single nucleotide in betaglobin.
Well that's 1 base pair.
So that means I'm trying to identify something that's on the order of 1/10e9
actually less than 1/10e9 to the whole human genome.
Carrying out purifications like that is really kind of, er, hard to imagine.
But the way it was done was by the invention of cloning.
Let me briefly overview the idea of cloning and then we'll dive into the details.
The idea of cloning was, the way to purify individual molecules
will just be to take the molecules and just dilute them
So there is only one of each molecule. That's very pure, isn't it?
The problem is not very much.
So you need a way to take a single copy of a molecule and then make many copies of it.
So purification is not hard.
You just dilute it down so you work with single molecules.
But then you need to copy it back again, again and again.
And no biochemical technique involves, say, fractionating of a cell and replicating some enzyme,
you know copying some enzymes. You can copy enzymes, but you can’t copy DNA.
That was the basis of it. So here's the way it goes.
The basic way you'll look at is take your DNA and cut your DNA of interest
maybe the human genome, into pieces at defined sites.
Then paste your DNA, which is more technically, ligate, the word we use,
to some other DNA, called a vector
So cut your DNA and paste your DNA.
Each piece of your, say human DNA, gets stuck to some piece of vector.
Insert this DNA into vectors that can replicate in bacteria
So I'm gonna actually take my piece of human DNA
and just not ligate it to any other piece of DNA
I am gonna take my human DNA
and I am gonna ligate it to a vector that has all of the machinery,
all of the ability to be copied in a bacteria.
Then what I'm going to do is that I am going to transform my DNA
into a host cell, a host bacteria cell. Transform means introduce. 
When we talk about transforming DNA when I'm talking about changing it .
It's the word that’s used for taking my DNA stuck into a vector and introducing it into a bacteria cell.
Ideally, each bacteria cell would carry one such DNA molecular.
And then what I wanna do is I wanna plate my cells and select those that carry human DNA.
So I am gonna put them on a petri plate
and I want only the bacteria that happen to have picked up an individual piece of human DNA to grow.
That's the trick. It's a very simple trick.
Take total human DNA, cut it up into pieces,
glue it to a vector that's able to be replicated in bacteria,
put the vectors into bacteria cells.
Every bacteria cell picks up no more than one vector.
You plate it out and you simply arrange so that the only cells that grow
are those who picked up the piece of human DNA.
And every one of these colonies is the descendent of
a single bacteria cell that picked up a single human molecule
but is obligingly coping that molecule for you again, again and again and again.
And thus you have what we refer to.
This whole collection here is called a library of clones.
This is called a recombinant library because every piece
of the human genome is somewhere in here.
You know this one here probably is actin
 and maybe this one here maybe is collagen 11
and that one there might be other betaglobin, OK?
Actually, when you look at the plate there is no way
 to tell but in principle they are all there.
So there will be this question how do we look at the library
and pull out what the right one is.
But somewhere in there should be a bacteria colony that has
pure betaglobin gene, the DNA for betaglobin.
Tomorrow, the next lecture will be about how you actually find it,
but today let's just build this library.
So our goal is to be able to build a library like this. So...
We have figure out how to cut DNA, paste DNA, vectors, etc, etc.
So that's what our subject will be today. Let's dive in it.
First cutting DNA. How do you cut DNA? Restriction enzymes, etc.
It turns out that the way you could cut DNA at particular places, is as follows.
Let me take a piece of DNA.
Here is a double-stranded piece of DNA.
We’ll go AGCTAGAATTCTTACC. Hydroxyl there 3' end.
Let's go back on the other strand. What do we have?
GGTAAGAATTCTAGCT. Hydroxyl there, 3' end.
There's my double-stranded piece of DNA.
It turns out that there exists an enzyme that recognizes
that exact sequence: GAATTC.
The enzyme goes by the name EcoRⅠ.
This protein, this enzyme scans along the DNA and finds this sequence: GAATTC
Actually on this strand. What about on the other strand.
What does it say? Same thing.
It's reverse palindrome. It's symmetric.
That's very good. And it turns out most restriction enzymes do that.
Ok, so what it does when it finds that
With the benefit of color chalk that has just shown up here.
It cleaves the DNA fragment like that.
And what it gives you then is a broken double strand with an overhang: TTAA.
5', 3'. 3',5'. This has a hydroxyl here. This has a phosphate there.
And this other fragment here: AATTCTTACC GGTA...oops! It stops there.
So what happens is... and this has a hydroxyl 5', 3'. 3', 5'.
I get two fragments of DNA that have been broken there and have an overhang.
The overhangs are complementary. Those two sequences match each other.
There is what is called a 5' overhang and they're complementary.
So we have complementary, that is, matching 5' overhangs.
This is called EcoRI because it's purified, this particular enzyme, from E.coli, strain R.
And it's the number 1 such enzyme that was purified.
So it's very simple nomenclature.
Now here is the question: why do bacteria have an enzyme like this?
There is some people feel that the reason is that this enzyme is here precisely
to allow molecular biologists to cut and paste DNA.
And this represents pressures on the part of revolution.
There is other who think that's less likely. Me among them.
How did anybody find this stuff?
Well, shaggy dog story.  I have to tell you the following shaggy dog story.
So this is a fun shaggy dog story
and it's a MIT shaggy dog story because it comes from the work of Salvador Luria
who is a very famous biologist who worked here at MIT.
So, Salvador Luria was studying bacteria phage.
Remember bacteria phage are viruses that infect bacteria.
So he was studying some bacteria phage
and he took his bacteria phage
and used it to infect a strain of bacteria, strain A.
And he also used it to infect a strain of bacteria, strain B.
So when he did that, what you do is you plate a lawn of your bacteria cells.
You kind of have a schmush of bacteria cells that you plate here with virus mixed in.
And where there is the virus, the virus grows,
replicates and either kills or slows down the growth of the cells.
So the bacteria cells grow everywhere else.
But where a viral particle landed,
there is an absence of bacteria cells and that hole in the lawn.
This whole thing is called a lawn of bacteria
and the holes in the lawn are called plaques.
So when he did this, he found that
when he did it in strain A, he got a bunch of plaques.
And when he did it in strain B, he didn't. No plaques.
So what’s your simple explanation for this?
Strain B is different somehow, it’s resistant to the virus.
The virus has come in to do various things.
Strain B isn’t compatible with the virus or something like that. No big deal.
So it's the resistant strain.
But occasionally, you get a plaque. Very occasionally, you have an occasional plaque.
So how can this be? I said the strain was resistant.
How can there be an occasional plaque?
Mutation. In? Could there be a mutation in the bacteria? Sorry.
Well, if there is a mutation in the bacteria,
there’d one bacterium that had the mutation.
It was now susceptible and will die.
But the lawn would kind of grow
‘cause the cells around wouldn't have the mutation.
So it’s probably not a mutation in the bacteria but what could it be?
Maybe mutation in the virus.
What if it's the mutation in the virus that was able to overcome the resistance?
So that's ok. So what this must be, is the existence of a resisting virus
i.e. the virus that can overcome the resistance of the bacteria.
So far perfectly normal, no problem.
Now let's do the following experiment.
Let's take this resistant virus and grow it again on strain A, and grow it on strain B.
What do you think is gonna happen when I grow it in strain A?
Lot of growth, lot of plaques.
Still grows on strain A. And now, what's gonna happen when I grow it on strain B?
If this was really a mutation that made it able to grow on strain B,
then now we get lots of plaques.
This has now gained the ability to grow on strain B.
And sure enough that's what happens. So there is nothing funky, yet.
But now suppose I take one of these resistant viruses that I isolate here on strain B.
I grow it again here on strain A. It grows. I grow it on strain B. I grows.
I take it again from strain B and I repeat this.
I'll still grow on strain A and still grow on strain B.
Let's take one of those from strain A.
It's the resistant one which we now just happen to grow on strain A.
And now let's grow it again on strain A versus on strain B.
And sure enough it continue to grow on strain A , no problem.
And we grow it on strain B and what should we get?
It should grow on strain B, right?
‘Cause it’s mutant virus.
And you know it gained the ability to grow on either we pass it through B and pass it through A ?
But the answer was nothing. No growth. How can that be?
We had a virus we agreed that was a mutant virus
that had picked up the ability to grow on strain B
And we demonstrated it now grows on either A or B.
We then reached it, and grabbed the copy of it here from strain A,
having grown on strain A, and we try it again and now it won't grow on strain B.
If this was a mutation, I mean maybe the mutation reverted. Right?
It was the reversion of the mutation.
It mutated back. Is that possible?
No! Come on. The chance of that all of the copies have mutated back.
Come on. That's , I mean, you can repeat this several times and this is always what happens.
What does that tell you about this mutation in the virus?
It can't be a mutation of the virus because if it was mutation,
it would be transmitted through.
But passing through strain A makes it lose its stability to grow on strain B.
But as long as you keep passing it through strain B, it can grow on strain B.
This is not your typical genetics.
So Salvador Luria loved this and he really worked out what was going on.
And somehow, so anyway they refer to this as strain B
having the ability to restrict the growth of the virus.
Strain B can restrict the growth of the virus.
That's where this word, restriction enzyme, come s from.     
What's really truly going on here underneath the shaggy dog story,
It took a long time before the shaggy dog story, that Salvador Luria
was going to really, demonstrate, was fully worked out.
But what turns out to be the case is that
strain B has a restriction enzyme that’s how it restricts the growth.
It has one of these enzymes that can cut DNA at a specific place.
When the virus comes in to strain B, it injects its DNA
and the enzyme comes along and cuts the virus' DNA, protecting the bacteria.
It’s got its own defense mechanism. Pretty cool. Pretty cool.
So any DNA that's introduced if it has the sequence here
, GAATTC, the bacteria cuts it.
Wait a second. The bacteria has its own DNA.
Why doesn't it chop up its own chromosome?
Yeah. Maybe, so you know,
one simple possibility would be that
if this thing is looking for the sequence, GAATTC, in the genome,
maybe it's the case that the bacteria has arranged its own DNA never has a GAATTC.
That'd be a simple solution, right?
But is it a plausible solution? Why not?
Well just statistically, how often do I expect to encounter a GAATTC?
What's the frequency of any given six-letter word in a four-letter alphabet?
It's about a 1/4^6. So one in 4^6 positions would be GAATTC. That's about 4000 letters.
So every 4000 letters I expect to encounter a GAATTC.
How big is the E.Coli genome? 4 million letters.
So how many GAATTC will there be? About 1000 of them.
It’s just not plausible to imagine it didn't have the sites?
So your idea is if it has these sites it got arrange to protect its own sites.
So how is it gonna protect the sites? Cover it with something or...
You could imagine something covers it or something. 
You wanna alter your own sites. It turns out, you are exactly right.
What happens here is that there is an enzyme that
comes along at this position attaches a methyl group.
It modifies the DNA, modifies the DNA by attaching a methyl group.
It turns out that methyl group is enough to prevent the restriction enzyme from binding.
So this blocks the restriction enzyme.
So that way the bacteria is able to distinguish between its own DNA,
which is methylated, and the viral DNA.
So wait a second, how did it explain that my virus that managed to grow?
How did my virus manage to grow?
It would need to get itself modified also to be protected.
Could that happen by chance? 
What if the methylation enzyme, the methylase,
which is floating around in the cell accidentally,
 quoting quotes, methylated the viruses' DNA?
What would happen then? The virus would become immune.
So suppose the bacteria is pretty clever and
it had a lot more restriction enzyme and only a little bit of methylase.
Well you’d imagine that most of the time
 the restriction enzyme would cut up the viral DNA first.
But every once in a while the methylase will get there first and protect the viruses' DNA.
That becomes an immune virus because it can't be cut by the enzyme any more.
And if I take that and I grow it again on strain B,
it will now produce lots of plaques because it was methylated.
And if I grow it again on strain B, and if I grow it again on strain B,
it remains methylated, ‘cause once it’s methylated and comes into the cell,
it is not cut and so the descendents will get methylated.
But what happens if I ever grow that virus, the methylated virus on strain A?
Strain A doesn't have the restriction enzyme and it doesn't have the methylase.
So the progeny phage that grew up on strain A aren't methylated.
They are no longer protected.
The protection that the virus has is the protection that comes from this methylation enzyme.
It's not the sequence of the DNA. It's the attachment to its methyl groups.
And so it turns out that if you ever pass this virus through strain A, passage through strain A,
the resulting DNA loses, is unmethylated and now it can be cut.
And it can be cut. Well, this explained the weird results of Luria. 
That's somehow bacteria has a complex defense mechanism of restriction enzyme and cognate methylase.
The restriction enzyme would cut the sequence.
The chromosome would be protected by methylating that site.
And usually we'll find occasionally of the bacteria virus would get methylated,
and would be protected as long as it continues to
go to strains that have this restriction-methylation system.
That was it. This shaggy dog story took a couple of decades to work out
and eventually leads the Nobel Prize for the discovery of the restriction enzymes.
They are extremely important because although bacteria do this to protect themselves,
they have also given us the perfect tools to now cut DNA where we want to cut DNA.
Now what if you wanted to cut the GAATTC? You've got EcoR I.
But what if you wanna cut another sequence?
Well, it turns out that if you wanna cut it at GGATCC, there is an enzyme called BemH I.
If you wanna cut it at AAGCTT, or AAGCTT there is an enzyme called Hind III.
If you wanna cut it at just GATC like this, CTAG there is an enzyme called Mbo I.
And there are enzymes that cut it this way, enzymes that cut it this way,
enzymes that cut it this way, enzymes that recognize 6 bases, 4 bases.
There are even enzymes that recognize 8 bases.
It turns out that bacteria have elaborated zillions of different
restriction enzymes that recognize different sequences.
This is perfect for molecular biologists.
bacteria, of course, being much smaller than we are, have been out for much longer,
have developed all of these tools for engineering.
All we have to do is borrow them.
So how do you get EcoR I? We grow that strain of E.Coli. You purify EcoR I.
And how do you get Hind III? You grow that strain of Haemuphilus Influenza.
You purify the enzyme.At least that's how primitive molecular biologists did.
If you wanted to work with restriction enzymes,
You’d grow up the bacteria, you’d purify the enzyme yourself.
And you'll just use it in your laboratory.
Of course today, what does a modern a molecular biologist do
if he or she should want Hind III?
It's in the catalog. So the catalog has 200 restriction enzymes.
Yeah, Psi I is a new on sale 500 units for 400 dollars.
See what's EcoR I is going for. 
See. EcoR I, look at it. 50,000 units for 200 bucks.
Good price for EcoR I. It’s a famous enzyme here.  
So all you have to do is you give them your credit card number
and you have it tomorrow by Fedex
So that's how restriction enzymes are obtained today.
So next up. We can cut DNA any place we want to.
We now need to glue DNA together.
Suppose I cut DNA, human DNA and I'm gonna cut it
or just take human DNA, your DNA which are purified.
And I am gonna cut it at all its EcoR I sites. I can take any other DNA I want.
I don’t know, I could take zebra DNA. I could take anything
 and I could also cut it at EcoR I sites. I could mix them together.
And after mixing them together the fragments would
 float around and remember this down here has TTAA.
This fragment over here from some other piece, TTAA.
This could be human DNA. This could be zebra DNA if you want to. It doesn't matter.
Could be bacteria DNA. These fragments overlap.
Their will hydrogen bond a little bit, but that
 of course won't introduce a covalent bond here.
I'd really like to make a covalent bond.
I'd like to attach the piece of DNA from one source
to the piece of DNA from the other source
by doing the opposite of the restriction enzyme.
Restrictions enzymes cut at these locations.
I would now like to catalyze the rejoining of the sugar-phosphate backbone here.
So I'd like to rejoin the sugar-phosphate backbone.
I have a hydroxyl here. I have a phosphate here.
And I would like to ligate them together.
So how do I manage to ligate?
What kind of fancy chemistry do I do to ligate these pieces of DNA together?
I don't do any fancy chemistry.
I again ********* of the bacteria who have solved all these problem for.
I ask bacteria: how do you do this?
And it says: well, we have an enzyme called ligase.
So you purify ligase from bacteria. You add that
and ligase ligate these fragments together.
Why do bacteria have an enzyme ligase?
For repair of their own DNA, things go wrong.
This is part of the DNA maintaining scheme of a bacteria
They have the enzyme ligase to repair their own breaks in DNA.
And obligingly you can purify DNA ligase.
So you have ligase. And today of course, if you need ligase, how do you get it?
To the catalog. Absolutely!
So you can glue together any of those things you want.
All right. Next up. What DNA do I wanna stick together?
I mean I here made a silly example.
I am gonna stick some human DNA to some zebra DNA. Why do that?
I am just to show you that I can do it, right?
I am just demonstrating that I can stick any DNA to any DNA.
Remember the DNA doesn’t… Once I’ve got a piece of DNA.
It isn't know whether it came from human or zebra. It's just a molecule.
You can stick the molecules together. Right?
But what do I really wanna attach my human DNA to?
I wanna attach it to some other DNA
that has the ability to grow on its own within bacteria.
Vectors. I need to make. Here's what I'd really like.
I would like to have a piece of DNA that has some sequences
that contain the recognition sites for replication.
I'd like to have some replication initiation sites here.
So a piece of DNA that,- remember this that the bacteria chromosome itself.
Here is my bacteria. The bacteria's own chromosome replicates itself.
And it has the ability to start DNA replication at multiple sites called origins of replication.
But what I'd really like is to be able to construct in a laboratory
a synthetic piece of DNA that also would function as an origin of replication.
'Cause then what I could do is in vitro take my piece of DNA, attach it to this vector.
And it would now have the ability to grow in bacteria.
How am I gonna make a piece of DNA?
What kind of engineering tricks can we do to create a small piece of DNA
that has all the machinery needed to be able to be copied, replicated just like bacteria chromosomes?
That's a pretty fancy field of engineering.
How are you gonna do that?
Sorry. Viruses. Who are you gonna ask?
If you wanna do this, you can ask the experts. Who are the experts?
Viruses or ? Or bacteria or phage?
Basically if you wanna do anything the place to ask is the folks have the most experience.
The folks have the most experience are almost always prokaryotic organisms
because they are by far the most evolved things on this planet.
Anything that can replicate itself and grow every 20 minutes or something like that.
It’s a lot more generations of evolution than you have.
And therefore they are much more optimized than we are.
So you go asking say: Have any bacteria worked out how to do this?
(It) turns out bacteria has worked out how to do this just fine.
In fact, most bacteria ,at least many bacteria, contain within them,
in addition to their own chromosome, small cycles of DNA.
These are called episomes. This is the chromosome.
Epi- means on top of or in addition to.
So in addition to the chromosome there is an episome.
The episome is in fact an autonomously replicating piece of DNA that has an origin.And it replicates.
Why do bacteria have episome?
It turns out episomes often contain genes.
One of the genes they contain or some of the types of genes they contain are resistance genes.
There might be, for example, an penicillin resistance gene contained on the episome.
Or streptomycin resistance gene.
It turns out the bacteria have these episomes containing resistance genes
 and they are not in the chromosome.
They are separate. Why would they do that?
It turns out when the bacterium dies, the cell cracks open, the DNA spills out.
The next door neighbor bacteria has mechanism to suck up DNA from the environment.
Never know, might find something interesting out there.
So it turns out that bacteria are rather promiscuously exchanging pieces of DNA all the time.
And so a bacteria that has a episome that has penicillin resistance gene can spread it to other bacteria.
And it's very nice. It's compact.
It's on its own little episome autonomously replicating piece of DNA.
This is great for bacteria wanting to spread drug resistance.
It's not good for human population.
For example, because this is how drug resistance spreads to populations.
This why we have spreads of penicillin resistance. Now, of course, wait a second.
This whole mechanism of spreading drug resistance.
We’ve only had antibiotics since the 1940s'. How did bacteria devise it so quickly?
Sorry. Many generations since 1945? That would be very impressive.
People ask why did they have this episome mechanism, the ability to spread DNA and all that.
That's an awful lot to evolve in 50 years?
Yep. Something natural like penicillin.
It turns out we didn't think of penicillin.
Who thought of penicillin? Fungi.
Right, again we learned from a lot of organisms.
Penicillin comes from fungi.
bacteria have be fighting of penicillin for millions and tens of millions of years.
We may be very proud of our penicillin and all that, but they've been at this for a very long time.
This is about war between bacteria and fungi. That's what this is, OK?
So that's why these things are here.
They are here so the bacteria can have these resistance genes against fungi and things like that,
 that make antibiotics, antibiotics are natural.
We've a few new ones, but most antibiotics have been made by nature.
And so if I wanted to replicate DNA,
if I wanted to attach my human DNA to a piece of DNA that’s capable of autonomous replication .
Autonomously replicating circles of DNA.
These autonomously replicating circles of DNA are also called plasmids.
That's the word we mostly use for them. Plasmids.
All I need to do is purify a plasmid from bacteria.
So I find the bacteria that has plasmids.
I purify the plasmid and then I can cut open the plasmid at the EcoR I site.
OK? So this plasmid would have an ORI, an origin of replication.
I cut it open at the EcoR I site. I'll take human DNA fragments that I've cut with EcoR I.
I'll mix them with plasmid DNA that has been opened up, has an origin.
Ligase will come along joining this up.
And now I have a cycle of DNA that has all the machinery to autonomously replicate plasmid DNA.
Now if I wanted get a vector, or honest-to-goodness plasmid,
I can go to a bacteria, grow it up, purify the plasmid and cut it.
Or alternatively if I need the plasmid, say tomorrow, it's in the catalog.
Next section of the catalog is long list of plasmids.
There is a plasmid there. All right, here's a nice plasmid.
Oh, yes. Let’s see pUK is a very good plasmid. pbr322 is a good palsmid.
Whole section, all this purple stuff is plasmids.
So you get the plasmid, too.
*******won't worry,
you get the restriction enzyme, you get the ligase, you get plasmids, no problem.
So I can then take total human DNA cut up, cut up, cut up, cut up.
Adding plasmid, and I am gonna ligate together.
And then having ligated my human DNA to my plasmids, I am going to mix with bacteria.
I take some bacteria cells, I add my mixture of these plasmids containing human DNA,
and now all I have to do is to persuade the bacteria to suck up my plasmids containing human DNA.
How do I teach bacteria to suck up DNA?
They do that for a living. That's what they do.
They are always spreading material. They have that ability.
All we’re doing is we’re using their ability.
You see, you get the sense that the kind of engineering that really works in biology
is engineering that exploits what nature has been doing for a very long time
rather than budding your head against the problem.
Usually somebody has solved it and it’s almost always bacteria.
So you transform the bacteria.
Now there are a few tricks you can use to make them a little more transformable.
You can add calcium phosphate and bla bla bla.
But anyway you can truly persuade them to take up the DNA.
And all you have to do is plate them out on a plate. Plate them out fairly dilutely
So there are a lot of single bacteria cells that land on the plate.
And wait for them to grow up.
Each one of these had a single plasmid, a different plasmid than the next guy over.
Wait a second. Each one?
How do I guarantee that every bacterium in my test tube took up a plasmid?
Is that possible? I mean I can't guarantee that every bacteria is gonna take up a plasmid.
Maybe I add so much plasmid that every bacteria will take one up.
That's a bad idea because why?
‘Cause then a lot of them will take more than one. You don't wanna to do that.
You really only wanna have at most one.
So if you're gonna arrange so that randomly one only have about one.
You gonna a lot that have zero. So this is a problem. It's the real waste.
My library is gonna have large numbers of bacteria that don't have any plasmid.
In fact, this transformation process is not so efficient.
Not so efficient. So we have a little bit of a problems here.
Some of these guys will have human DNA, but most of them won't.
So what can I do to arrange that any bacteria
that did not pick up a plasmid was incapable of growing?
Yep. Woooo, add a resistance gene to the plasmid.
Suppose I was so clever to add to that plasmid penicillin resistance.
So not just an origin of replication.
But suppose I also had a resistance gene here, say for penicillin resistance.
Or streptomycin resistance or ampicillin tends to be a big favor. ampicillin resistance.
Then my plasmid would have ampicillin resistance gene,
encoded on it an enzyme that can break down ampicillin.
And what do I do to my petri plate? I just add ampicillin.
Now even though most of the bacteria have not picked up a plasmid,
only those bacteria that have picked up a plasmid,
have the ampicillin resistance gene and can grow on an ampicillin containing plate.
How do I get a plasmid containing the ampicillin resistance gene?
That's in the catalog. It's all there. Right?
In fact, these occur naturally you can , you know,
with restriction enzymes move the ampicillin resistance gene to your favored plasmid.
If you don't like that, you could put it in kanamycin resistance, etc, etc, etc
So that's how you do that. So we've got the big picture here. We have now gotten a library.
The library of human fragments contained in E.Coli.
The library, a big petri plate or many petri plates.
Each one of which is a colony.
Each colony has a single vector with an origin,
 a resistance mark and a distinct piece of human DNA.
In this library lives somewhere the gene for Huntingdon's disease.
Over here a gene for cystic fibrosis.
So over here a gene for Duchenne muscular dystrophy.
Over here a gene for diastrophic dysplasia. Over here a gene for etc, etc.
The only detail now, you’ve got a library.
You've managed to purify each piece of
human DNA away from every other piece of human DNA.
The only question now is how do you use the library?
How do you go to the library and withdraw the correct volume from the shelf?
How do you find the one you are looking for?
So we have converted the problem of purification
Which in every other form of biochemistry starts by saying
 I’m gonna purified something based on its distinctive properties
to I'm gonna randomly purify everything. Everything will be purified in its own bacteria.
And now I've converted the problem of finding the one that I want in my library.
Next time we'll talk about how you go to the library and find which you want.
See you then.