PROFESSOR: Recombinant DNA, often referred to also as
genetic engineering.
This is a series of techniques, series of methods
that allow us to manipulate DNA for a variety of reasons.
Now, we take it for granted.
It's very much part of our everyday life in the
laboratory.
It's made a huge impact on the biotechnology and
pharmaceutical industries as well.
It wasn't always so uncontroversial.
In fact, in the 1960s and '70s, when this technology was
first being developed, it was great concern about scientists
manipulating DNA, manipulating genetic material.
In the city of Cambridge, in fact, had a moratorium for a
while on the practice of genetic engineering or
recombinant DNA technology, which fortunately, ultimately
was overcome with good practices and I think good
education about what the real limits of risk and benefit
were so that now it's used very widely and, I would also
say, very safely.
The bottom line for what we're going to talk about today and
for this section of the class is the ability to isolate and
to amplify specific DNA sequences.
They might be particular genes of interest to us.
They might be whole genomes.
They might be other regions of DNA, but we need to be able to
isolate them from the cells of the organisms of interest,
amplify them up into large quantities in order to be able
to study them in detail, and to make modifications in them.
This is done in a variety of organisms for
a variety of purposes.
One of them, and this is by no means the only one, but one
that sort of strikes home is the ability to make
therapeutic proteins, to be able to manufacture in the
laboratory or in a company proteins they could have
benefit for patients who are, for example, lacking the
function of a particular protein or enzyme leading to a
disease state.
One can use these methods to produce that protein in the
laboratory, and treat the individual.
This can be done in bacteria such as E. coli.
E. coli, which is the common gut bacterium that we all
carry, a very useful organism.
We use it in the lab.
We're going to use in our demonstrations today.
This is the standard vehicle in which we'd grow up,
recombinant DNA, amplify recombinant DNA for further
uses, but we can also make therapeutic proteins using the
manufacturing capabilities of the bacterium.
Plants, likewise, can be a source
of therapeutic proteins.
We can modify the genomes of plants so they will produce in
large quantities therapeutic proteins of interest, which
then can be ingested by the individual, which can reduce
production costs significantly,
and animals, likewise.
We can manipulate the genes of animals so that they will
express a protein of interest and secrete that protein, for
example, into the milk.
So there are so-called transgenic cows and transgenic
goats that produce therapeutic proteins in the mammary gland
and secrete those therapeutic proteins into the milk.
So the individual just needs to drink the milk and receive
the relevant dose.
So there are lots of ways, that this technology can be
helpful including in the context of medicine.
We can also engineer organisms.
I've already given you examples of that, but that was
really for the purposes of using those organisms as a
factory to make something of interest to us.
But we can manipulate the genes of plants, for example,
to make them resistant to various pests to make them
more robust, to give them a longer shelf life.
We can manipulate them for the better production of things
that are valuable to us.
Again, this is a bit of a controversial area,
genetically modified foods, not always well-accepted by
everyone because again, the thought is, this might be
disrupting the food chain in important ways, and this might
be ultimately not so beneficial.
Personally don't agree with that, but lots of people do
feel that way.
We're going to teach you the methods that we use to allow
us to do this.
And again, in work that I've alluded to from my own lab, we
use these methods to manipulate the genes of
animals, in our case it's mice, but there are lots of
animal species that one can use in order to create, for
example, disease models.
We talked to you about, Professor Brown talked to you
about genetic diseases.
They have specific alterations in genes.
We can use these methods to make similar alterations in
the genes of mice and other animal species and then study
the disease process in those animals, develop new
treatments in those animals in ways that are
hard to do in people.
So there's lots and lots, and these are just a few examples,
lots and lots of important uses for genetic engineering
and recombinant DNA technology.
One that we'll emphasize in just a few lectures and is
becoming extremely common and popular nowadays is DNA
sequencing.
You need to isolate the DNA from the organism of interest
and then have it in such a fashion that you can sequence
it's nucleotides.
This is a very popular activity now.
And specifically with respect to human health, DNA
sequencing, But some of the other methods that we're going
to talk about as well, allow us to characterize
disease-causing mutations at molecular detail.
So we know what causes x disease or y disease.
We can understand the consequences for the
individual-encoded proteins for the pathways that they
regulate, and we can come up with better medicines.
I think about this with respect to cancer, but it's
really changing the treatment of all diseases as we
understand more the molecular basis, the genetic basis of
those diseases.
And these techniques have been essential to
allow us to do that.
I want to cover a little bit of history so that you know
from whence this came.
Things really began to pick up in this
field in the late 1960s.
And the critical advance at this stage, was the discovery
of a method to cleave DNA into defined fragments, to start
with a genome or chromosome and to be able to cut it
particular places reproducibly so that one could isolate
fragments of DNA away from the mass of DNA, isolate a
particular region of the DNA away from everything else
using this method.
It wasn't enough just to cut the DNA up.
You had to amplify it up.
In order to amplify it up, you needed a vessel in which to do
the amplification, and the vessel of choice as I
mentioned was bacteria.
And this relied on a method that actually was known for
decades before but actually was not used for this purpose
until the early 1970s, and it's called bacterial
transformation.
You can transform bacteria by adding a new DNA sequence, and
the bacteria will take up that new DNA sequence and begin to
express the genes that are present on that DNA sequence
as if it was one of their own.
So the transfer of DNA a was critical, this process of
bacterial transformation.
And then the final thing which also occurred in the 1970s was
the identification of methods to amplify DNA sequences once
they got inside of bacteria.
It wasn't enough to actually get them in there.
You needed some special way to cause the bacterium to
amplify, that is, to replicate the DNA that was present
within them.
And these three events, which all came together in about a
five to ten year period, really initiated, launched
what we now call the recombinant DNA revolution and
initiated the biotechnology industry, which
started in the mid 1970s.
And an individual above all others who is credited with
launching the biotechnology industry was Robert Swanson
who in 1966 or so was sitting where you
are as an MIT freshman.
Bob Swanson was class of '69, actually.
And at the age of 28, in 1976, founded the company Genentech
with a scientist, Herb Boyer, who was very instrumental in
some of those breakthroughs that I just mentioned to you.
And I mention Bob to you now because of your connections
through MIT.
But he was a wonderful guy and sadly passed away from
glioblastoma at the age of 52.
So he didn't really get to fully realize the benefits of
what he had started, but he did, in fact,
start a great deal.
And this is a famous statue at Genentech which shows Herb
Boyer and Bob Swanson talking about the idea for the first
time and you might notice over a beer, very important part of
science, discussions over a beer.
In addition to this connection to MIT and really in honor of
Bob's connections to MIT, and a great pride that Bob had
actually in MIT, credited MIT greatly with him learning
about science, the importance of science, and also business.
He got a degree in chemistry as well as a degree from the
Sloan School.
We decided when we launched the Koch Institute to name a
very large space in the Koch Institute for Bob Swanson.
It's called the Swanson Biotechnology Center.
You can visit it.
So it's a series of core facilities that support all of
our researchers.
And indeed, researchers across the MIT campus, and this is a
nice quote from Bob's widow, Judy Swanson, who has been
very supportive of this effort.
So Bob Swanson and MIT, in many ways, actually, have a
lot to do with the technology and the revolution that we're
going to talk about now.
All right.
So as I mentioned, we are going to do a demo.
We're going to teach you a real-life example now of how
this work is done.
And our goal is to clone a gene.
You can actually wait right there, Anna.
Thanks.
We're going to clone a gene, and we're going to clone a
particular gene.
It's a toxin gene.
It's a gene from a pathogenic bacterium.
And you might ask the question, a reasonable
question, why the heck would you do that?
Why would you clone a toxin gene?
So what do you think?
What's the purpose in the laboratory of cloning out a
toxin-encoding gene from a pathogenic bacterium?
So several people have suggested the obvious was that
you want to engage in global terror, which we actually
don't support here at MIT, so we're going to take that down.
But maybe somebody else is going to do that.
So perhaps it would be good if we got one step ahead of the
game, isolated the gene, manufactured the protein, and
then made a vaccine against that toxin so that we could
prevent the bad consequences of exposure to the toxin.
Or maybe that thing is actually very interesting
independent of its bad uses.
We could learn stuff, which might be helpful ultimately in
related activities.
So for the general purpose of biomedical research, we often
study how these organisms work because it can teach us
things, sometimes surprising things that are
useful down the road.
The organism in question is Streptococcus pyogenes.
Streptococcus pyogenes, which causes in certain cases, in
certain individuals a disease called necrotizing fasciitis.
Necrotizing fasciitis, which is otherwise more commonly
called the flesh-eating disease.
And you might think I'm joking, but I'm not.
This is a true thing.
This is a true thing, and some of you might be squeamish, and
if you are, and I'm being serious here.
If you're squeamish looking at ugly, nasty, disgusting
pictures, close your eyes for a second.
I'll tell you when you can open them.
But this is an individual who was exposed to this bacterium
and developed necrotizing fasciitis.
That's a real-world case, so it is really pretty bad.
You can open your eyes now if you closed them.
I hesitate to show that slide because in the past, I've had
a few boys throw up when I showed that slide.
So what are we going to do?
Well, we're going to isolate this gene.
And in order to isolate this gene, we need to be able to
separate it, this gene, from the chromosome
in which it is contained.
And the chromosome from which it is contained is the
chromosome of S. pyogenes.
So this is the S. pyogenes chromosome.
It's about four million base pairs and it
has about 1,000 genes.
So spread throughout this circular chromosome, there are
lots of genes.
We're interested in one of them.
Now, this chromosome also has another thing on it, which I
hope you all know about from the material that Professor
Sive just covered for you.
What is the one piece of DNA material that all chromosomes
need in order to replicate?
AUDIENCE: Origin of replication.
PROFESSOR: An origin of replication, very good.
An origin of replication.
It has an origin of replication, and then it has a
bunch of genes.
It has gene A. I'm just making this up.
It has gene B, and it has a whole bunch of
other genes as well.
And then it has -- and imagine that this orange chalk was red
because it's more effective if it's red.
It has the T gene, and that's the toxin gene.
So our goal is to transfer the T gene and not the rest of
this stuff, because we don't actually care about the rest
of the stuff.
We just care about the T gene --
into the E. coli cells for the reasons that I
mentioned up there.
And in order to do that, we have to grow large amounts of
the organism that's going to in a sense donate the DNA.
We then isolate the chromosomal DNA, and we'll
show you how.
We're then going to use this method to fragment the DNA not
randomly, but in specific places.
And then we're going to transfer the DNA of interest
to E. coli using this method of transformation.
We're going to take this fragment and move it through
the membrane of the E. coli so that it becomes resident
inside the E. coli cell.
So now on to our demonstration and my lab assistant, Anna
Deconinck, will help me here.
So what we've done is to, in the laboratory, isolate S.
pyogenes as well as E. coli, grow them up in large
quantities.
You've got your gloves, right?
So I'll take the buffers.
So we have various solutions and buffers that will allow us
to sort of wash the stuff we don't want away from the
bacterial cells, lice the bacterial membrane, isolate
the nucleic acid away from all the other stuff that's inside
the cells, and then we'll purify the chromosomal DNA.
So Anna has grown up E. coli and S. pyogenes, taken that
suspension of cells, and used a centrifuge to spin those
cells down to the bottom of these tubes here-- you can
show them, Anna-- these tubes here.
And the first thing we need to do is get rid of the
supernate, the broth that the cells grew in.
So first, we're going to decant.
Here, you can decant the pyogenes, but be
careful with it.
So we're going to decant this in order to grow up the
amounts of bacteria that we need.
Now we're doing this in a very small quantities.
In fact, in industrial scale, you do it in huge--
ANNA: Sorry.
PROFESSOR: Ah, that's a problem.
It's actually a bit more of a problem.
ANNA: I just have a buffer to wash.
PROFESSOR: I don't think that's going to do it, Anna.
Dude, we may need to actually skip, we may need to cancel.
This is a little more serious.
Wait a minute.
Well, I don't know.
Maybe.
Let's just see if it's safe or not.
I think it'll be okay.
All right.
That was a joke.
She did very well though, don't you think?
She did very well.
That was outstanding.
Anybody want some apple juice?
You're welcome to it.
ANNA: It needs ice.
PROFESSOR: Of course we would never bring pathogenic
bacterium to class.