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PROFESSOR: Recombinant DNA,
often referred to also as

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genetic engineering.

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This is a series of techniques,
series of methods

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that allow us to manipulate DNA
for a variety of reasons.

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Now, we take it for granted.

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It's very much part of our
everyday life in the

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laboratory.

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It's made a huge impact on
the biotechnology and

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pharmaceutical industries
as well.

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It wasn't always so
uncontroversial.

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In fact, in the 1960s and '70s,
when this technology was

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first being developed, it was
great concern about scientists

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manipulating DNA, manipulating
genetic material.

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In the city of Cambridge, in
fact, had a moratorium for a

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while on the practice of
genetic engineering or

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recombinant DNA technology,
which fortunately, ultimately

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was overcome with good practices
and I think good

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education about what the real
limits of risk and benefit

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were so that now it's used very
widely and, I would also

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say, very safely.

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The bottom line for what we're
going to talk about today and

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for this section of the class is
the ability to isolate and

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to amplify specific
DNA sequences.

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They might be particular genes
of interest to us.

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They might be whole genomes.

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They might be other regions of
DNA, but we need to be able to

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isolate them from the cells of
the organisms of interest,

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amplify them up into large
quantities in order to be able

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to study them in detail, and to
make modifications in them.

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This is done in a variety
of organisms for

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a variety of purposes.

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One of them, and this is by no
means the only one, but one

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that sort of strikes home
is the ability to make

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therapeutic proteins, to be
able to manufacture in the

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laboratory or in a company
proteins they could have

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benefit for patients who are,
for example, lacking the

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function of a particular protein
or enzyme leading to a

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disease state.

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One can use these methods to
produce that protein in the

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laboratory, and treat
the individual.

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This can be done in bacteria
such as E. coli.

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E. coli, which is the common
gut bacterium that we all

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carry, a very useful organism.

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We use it in the lab.

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We're going to use in our
demonstrations today.

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This is the standard vehicle
in which we'd grow up,

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recombinant DNA, amplify
recombinant DNA for further

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uses, but we can also make
therapeutic proteins using the

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manufacturing capabilities
of the bacterium.

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Plants, likewise,
can be a source

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of therapeutic proteins.

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We can modify the genomes of
plants so they will produce in

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large quantities therapeutic
proteins of interest, which

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then can be ingested by the
individual, which can reduce

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production costs significantly,

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and animals, likewise.

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We can manipulate the genes of
animals so that they will

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express a protein of interest
and secrete that protein, for

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example, into the milk.

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So there are so-called
transgenic cows and transgenic

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goats that produce therapeutic
proteins in the mammary gland

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and secrete those therapeutic
proteins into the milk.

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So the individual just needs to
drink the milk and receive

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the relevant dose.

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So there are lots of ways, that
this technology can be

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helpful including in the
context of medicine.

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We can also engineer
organisms.

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I've already given you examples
of that, but that was

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really for the purposes of using
those organisms as a

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factory to make something
of interest to us.

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But we can manipulate the genes
of plants, for example,

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to make them resistant to
various pests to make them

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more robust, to give them
a longer shelf life.

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We can manipulate them for the
better production of things

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that are valuable to us.

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Again, this is a bit of
a controversial area,

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genetically modified foods, not
always well-accepted by

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everyone because again, the
thought is, this might be

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disrupting the food chain in
important ways, and this might

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be ultimately not
so beneficial.

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Personally don't agree with
that, but lots of people do

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feel that way.

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We're going to teach you the
methods that we use to allow

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us to do this.

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And again, in work that I've
alluded to from my own lab, we

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use these methods to manipulate
the genes of

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animals, in our case it's mice,
but there are lots of

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animal species that one can use
in order to create, for

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example, disease models.

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We talked to you about,
Professor Brown talked to you

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about genetic diseases.

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They have specific alterations
in genes.

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We can use these methods to make
similar alterations in

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the genes of mice and other
animal species and then study

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the disease process in those
animals, develop new

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treatments in those animals
in ways that are

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hard to do in people.

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So there's lots and lots, and
these are just a few examples,

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lots and lots of important uses
for genetic engineering

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and recombinant DNA
technology.

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One that we'll emphasize in
just a few lectures and is

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becoming extremely common and
popular nowadays is DNA

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sequencing.

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You need to isolate the DNA from
the organism of interest

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and then have it in such a
fashion that you can sequence

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it's nucleotides.

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This is a very popular
activity now.

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And specifically with respect
to human health, DNA

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sequencing, But some of the
other methods that we're going

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to talk about as well, allow
us to characterize

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disease-causing mutations
at molecular detail.

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So we know what causes x
disease or y disease.

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We can understand the
consequences for the

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individual-encoded proteins
for the pathways that they

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regulate, and we can come up
with better medicines.

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I think about this with respect
to cancer, but it's

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really changing the treatment
of all diseases as we

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understand more the molecular
basis, the genetic basis of

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those diseases.

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And these techniques have
been essential to

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allow us to do that.

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I want to cover a little bit
of history so that you know

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from whence this came.

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Things really began
to pick up in this

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field in the late 1960s.

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And the critical advance at this
stage, was the discovery

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of a method to cleave DNA into
defined fragments, to start

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with a genome or chromosome
and to be able to cut it

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particular places reproducibly
so that one could isolate

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fragments of DNA away from the
mass of DNA, isolate a

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particular region of the DNA
away from everything else

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using this method.

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It wasn't enough just
to cut the DNA up.

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You had to amplify it up.

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In order to amplify it up, you
needed a vessel in which to do

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the amplification, and the
vessel of choice as I

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mentioned was bacteria.

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And this relied on a method that
actually was known for

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decades before but actually was
not used for this purpose

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until the early 1970s, and
it's called bacterial

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transformation.

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You can transform bacteria by
adding a new DNA sequence, and

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the bacteria will take up that
new DNA sequence and begin to

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express the genes that are
present on that DNA sequence

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as if it was one of their own.

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So the transfer of DNA a was
critical, this process of

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bacterial transformation.

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And then the final thing which
also occurred in the 1970s was

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the identification of methods to
amplify DNA sequences once

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they got inside of bacteria.

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It wasn't enough to actually
get them in there.

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You needed some special way
to cause the bacterium to

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amplify, that is, to replicate
the DNA that was present

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within them.

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And these three events, which
all came together in about a

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five to ten year period, really
initiated, launched

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what we now call the recombinant
DNA revolution and

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initiated the biotechnology
industry, which

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started in the mid 1970s.

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And an individual above all
others who is credited with

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launching the biotechnology
industry was Robert Swanson

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who in 1966 or so was
sitting where you

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are as an MIT freshman.

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Bob Swanson was class
of '69, actually.

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And at the age of 28, in 1976,
founded the company Genentech

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with a scientist, Herb Boyer,
who was very instrumental in

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some of those breakthroughs that
I just mentioned to you.

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And I mention Bob to you now
because of your connections

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through MIT.

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But he was a wonderful guy and
sadly passed away from

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glioblastoma at the age of 52.

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So he didn't really get to fully
realize the benefits of

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what he had started, but
he did, in fact,

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start a great deal.

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And this is a famous statue at
Genentech which shows Herb

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Boyer and Bob Swanson talking
about the idea for the first

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time and you might notice over a
beer, very important part of

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science, discussions
over a beer.

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In addition to this connection
to MIT and really in honor of

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Bob's connections to MIT, and
a great pride that Bob had

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actually in MIT, credited MIT
greatly with him learning

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about science, the importance of
science, and also business.

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He got a degree in chemistry as
well as a degree from the

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Sloan School.

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We decided when we launched the
Koch Institute to name a

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very large space in the Koch
Institute for Bob Swanson.

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It's called the Swanson
Biotechnology Center.

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You can visit it.

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So it's a series of core
facilities that support all of

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our researchers.

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And indeed, researchers across
the MIT campus, and this is a

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nice quote from Bob's widow,
Judy Swanson, who has been

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very supportive of
this effort.

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So Bob Swanson and MIT, in many
ways, actually, have a

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lot to do with the technology
and the revolution that we're

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going to talk about now.

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All right.

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So as I mentioned, we are
going to do a demo.

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We're going to teach you a
real-life example now of how

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this work is done.

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And our goal is to
clone a gene.

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You can actually wait
right there, Anna.

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Thanks.

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We're going to clone a gene,
and we're going to clone a

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particular gene.

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It's a toxin gene.

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It's a gene from a pathogenic
bacterium.

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And you might ask the question,
a reasonable

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question, why the heck
would you do that?

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Why would you clone
a toxin gene?

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So what do you think?

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What's the purpose in the
laboratory of cloning out a

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toxin-encoding gene from
a pathogenic bacterium?

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So several people have suggested
the obvious was that

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you want to engage in global
terror, which we actually

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don't support here at MIT, so
we're going to take that down.

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But maybe somebody else
is going to do that.

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So perhaps it would be good if
we got one step ahead of the

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game, isolated the gene,
manufactured the protein, and

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then made a vaccine against that
toxin so that we could

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prevent the bad consequences
of exposure to the toxin.

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Or maybe that thing is actually
very interesting

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independent of its bad uses.

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We could learn stuff, which
might be helpful ultimately in

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related activities.

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So for the general purpose of
biomedical research, we often

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study how these organisms work
because it can teach us

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things, sometimes surprising
things that are

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useful down the road.

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The organism in question is
Streptococcus pyogenes.

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Streptococcus pyogenes, which
causes in certain cases, in

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certain individuals a disease
called necrotizing fasciitis.

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Necrotizing fasciitis, which
is otherwise more commonly

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called the flesh-eating
disease.

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And you might think I'm
joking, but I'm not.

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This is a true thing.

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This is a true thing, and some
of you might be squeamish, and

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if you are, and I'm being
serious here.

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If you're squeamish looking
at ugly, nasty, disgusting

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pictures, close your
eyes for a second.

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I'll tell you when you
can open them.

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But this is an individual who
was exposed to this bacterium

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and developed necrotizing
fasciitis.

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That's a real-world case, so
it is really pretty bad.

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You can open your eyes now
if you closed them.

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I hesitate to show that slide
because in the past, I've had

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a few boys throw up when
I showed that slide.

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00:16:08,050 --> 00:16:09,090
So what are we going to do?

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Well, we're going to
isolate this gene.

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00:16:11,610 --> 00:16:15,010
And in order to isolate this
gene, we need to be able to

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separate it, this gene,
from the chromosome

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in which it is contained.

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And the chromosome from which
it is contained is the

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chromosome of S. pyogenes.

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00:16:34,660 --> 00:16:36,500
So this is the S. pyogenes
chromosome.

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It's about four million
base pairs and it

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has about 1,000 genes.

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So spread throughout this
circular chromosome, there are

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00:16:55,090 --> 00:16:56,030
lots of genes.

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We're interested
in one of them.

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Now, this chromosome also has
another thing on it, which I

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hope you all know about from
the material that Professor

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Sive just covered for you.

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00:17:05,760 --> 00:17:09,420
What is the one piece of DNA
material that all chromosomes

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need in order to replicate?

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AUDIENCE: Origin
of replication.

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PROFESSOR: An origin of
replication, very good.

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An origin of replication.

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It has an origin of replication,
and then it has a

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bunch of genes.

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It has gene A. I'm just
making this up.

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It has gene B, and it
has a whole bunch of

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other genes as well.

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And then it has -- and imagine
that this orange chalk was red

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because it's more effective
if it's red.

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It has the T gene, and that's
the toxin gene.

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So our goal is to transfer the
T gene and not the rest of

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this stuff, because we don't
actually care about the rest

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of the stuff.

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We just care about
the T gene --

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into the E. coli cells for
the reasons that I

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00:18:08,940 --> 00:18:11,320
mentioned up there.

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And in order to do that, we have
to grow large amounts of

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the organism that's going to
in a sense donate the DNA.

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We then isolate the chromosomal
DNA, and we'll

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show you how.

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00:18:41,730 --> 00:18:48,750
We're then going to use this
method to fragment the DNA not

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randomly, but in specific
places.

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00:18:56,280 --> 00:19:02,310
And then we're going to transfer
the DNA of interest

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to E. coli using this method
of transformation.

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00:19:06,580 --> 00:19:08,910
We're going to take this
fragment and move it through

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00:19:08,910 --> 00:19:11,700
the membrane of the E. coli so
that it becomes resident

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inside the E. coli cell.

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So now on to our demonstration
and my lab assistant, Anna

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00:19:19,120 --> 00:19:20,850
Deconinck, will help me here.

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00:19:20,850 --> 00:19:28,530
So what we've done is to, in
the laboratory, isolate S.

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00:19:28,530 --> 00:19:35,950
pyogenes as well as E. coli,
grow them up in large

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00:19:35,950 --> 00:19:36,900
quantities.

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00:19:36,900 --> 00:19:38,150
You've got your gloves, right?

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00:19:45,260 --> 00:19:48,510
So I'll take the buffers.

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00:19:48,510 --> 00:19:51,300
So we have various solutions and
buffers that will allow us

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00:19:51,300 --> 00:19:53,660
to sort of wash the stuff we
don't want away from the

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00:19:53,660 --> 00:19:56,980
bacterial cells, lice the
bacterial membrane, isolate

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00:19:56,980 --> 00:19:59,600
the nucleic acid away from all
the other stuff that's inside

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00:19:59,600 --> 00:20:07,720
the cells, and then we'll purify
the chromosomal DNA.

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00:20:07,720 --> 00:20:16,250
So Anna has grown up E. coli
and S. pyogenes, taken that

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00:20:16,250 --> 00:20:19,910
suspension of cells, and used
a centrifuge to spin those

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00:20:19,910 --> 00:20:22,720
cells down to the bottom of
these tubes here-- you can

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00:20:22,720 --> 00:20:24,360
show them, Anna-- these
tubes here.

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00:20:24,360 --> 00:20:27,400
And the first thing we need
to do is get rid of the

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00:20:27,400 --> 00:20:29,640
supernate, the broth that
the cells grew in.

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00:20:29,640 --> 00:20:31,740
So first, we're going
to decant.

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00:20:31,740 --> 00:20:34,580
Here, you can decant the
pyogenes, but be

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00:20:34,580 --> 00:20:35,310
careful with it.

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00:20:35,310 --> 00:20:39,220
So we're going to decant this
in order to grow up the

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00:20:39,220 --> 00:20:41,980
amounts of bacteria
that we need.

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00:20:41,980 --> 00:20:43,860
Now we're doing this in a
very small quantities.

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00:20:43,860 --> 00:20:46,740
In fact, in industrial scale,
you do it in huge--

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00:20:55,206 --> 00:20:56,456
ANNA: Sorry.

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00:20:59,688 --> 00:21:01,870
PROFESSOR: Ah, that's
a problem.

321
00:21:05,490 --> 00:21:07,401
It's actually a bit
more of a problem.

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00:21:07,401 --> 00:21:11,170
ANNA: I just have a
buffer to wash.

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00:21:11,170 --> 00:21:13,120
PROFESSOR: I don't think that's
going to do it, Anna.

324
00:21:13,120 --> 00:21:16,380
Dude, we may need to actually
skip, we may need to cancel.

325
00:21:16,380 --> 00:21:17,600
This is a little more serious.

326
00:21:17,600 --> 00:21:18,850
Wait a minute.

327
00:21:22,960 --> 00:21:23,460
Well, I don't know.

328
00:21:23,460 --> 00:21:24,200
Maybe.

329
00:21:24,200 --> 00:21:28,050
Let's just see if it's
safe or not.

330
00:21:28,050 --> 00:21:29,740
I think it'll be okay.

331
00:21:29,740 --> 00:21:30,210
All right.

332
00:21:30,210 --> 00:21:31,460
That was a joke.

333
00:21:35,480 --> 00:21:37,260
She did very well though,
don't you think?

334
00:21:37,260 --> 00:21:38,360
She did very well.

335
00:21:38,360 --> 00:21:39,695
That was outstanding.

336
00:21:49,730 --> 00:21:51,390
Anybody want some apple juice?

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00:21:51,390 --> 00:21:52,580
You're welcome to it.

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00:21:52,580 --> 00:21:55,166
ANNA: It needs ice.

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00:21:55,166 --> 00:21:58,430
PROFESSOR: Of course we would
never bring pathogenic

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00:21:58,430 --> 00:21:59,680
bacterium to class.