Overview of Recombinant DNA | MIT 7.01SC Fundamentals of Biology


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.

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