cDNA Libraries and Expression Libraries | MIT 7.01SC Fundamentals of Biology

cDNA Libraries and Expression Libraries | MIT 7.01SC Fundamentals of Biology


ERIC LANDER: That worked. Let’s do it again. Now, number 2. I would like to clone not the
Arg1 gene, but let’s say the beta globin gene from human. So let’s take human
beta globin. I want to clone it. So human beta globin,
hemoglobin, it’s a tetromer. It has four parts. It’s got an alpha, an alpha,
a beta, and a beta. Four proteins come together
in a protein tetromer. And it’s got two alphas,
two betas. The beta subunit of hemoglobin
is encoded by the human beta globin gene. That’s the nomenclature here. All right. Let’s clone beta globin. Same deal. How are we going to do it? Any takers? What should we start with? Yes? AUDIENCE: Find a restriction
enzyme? ERIC LANDER: Find a restriction
enzyme. I’ll go to catalogue and
try EcoRI today. OK? Now what? AUDIENCE: Add it to a bunch
of [INAUDIBLE]. ERIC LANDER: So I’m going
to start with human DNA? AUDIENCE: Yes. ERIC LANDER: Yes is
a good answer. Yes. I’m going to start
with human DNA. Good. Let’s get that up there. We’re going to start– AUDIENCE: [INAUDIBLE]. ERIC LANDER: Sorry? AUDIENCE: [INAUDIBLE]. ERIC LANDER: So we’ll start
with human DNA. There we go, pieces of human
DNA cut with EcoRI. Now, what are we going to do? Takers? Let’s attach it to a vector. Actually, wait a second. This vector here, I told you
that there were vectors that could grow in E. coli. Did I ever tell you there
were vectors that could grow a yeast? There are. And they’re in the catalog. OK. Yes? Now, what am I going to do? I’m going to attach
this to a vector. Now, what do I do? What vector do I want
to attach this to? Where is it going to grow? Human cells. That’s interesting. So one option would
be human cells. So we want to take a vector
that grows in human cells. Ha. Yep. AUDIENCE: [INAUDIBLE]. ERIC LANDER: Then I need
a mammalian origin of replication. AUDIENCE: [INAUDIBLE]. ERIC LANDER: And a mammal– Well, so it turns out that I
can technically do this. There are now vectors
that I can use to grow in human cells. It’s a lot harder to do. So let’s try doing it with a
microbe first, but then we’ll come back and we’ll
do it with human. So try doing this
in a microbe. What have you got
for a microbe? How do we do this
in a microbe? Well, let’s try E. coli for
the sake of argument. But we could also try, keep in
mind your human for a second. We do this, we do this,
we get a plate. One of these guys is going to
have alpha globin, one of them will have collagen. That guy here has beta globin. How are we going to
recognize it? This is tough. What were you going to
do for the human? AUDIENCE: No idea. ERIC LANDER: But you were
going to get it into a human cell. AUDIENCE: Yeah. ERIC LANDER: Were you going to
try to do complementation? AUDIENCE: Maybe. ERIC LANDER: Yeah. The problem doing
complementation is most human cells don’t need beta globin. What’s beta globin good for? AUDIENCE: [INAUDIBLE]. ERIC LANDER: What’s hemoglobin
good for? AUDIENCE: [INAUDIBLE]. ERIC LANDER: It’s in your red
blood cells to transport oxygen around. You think individual
cells care? Nah. Your marrow might,
but only in C2. The problem with doing it in a
human cell initially is, if we tried the same exact trick
and we tried a beta globin-deficient human cell,
and we’re going to try to complement with a beta globin,
the problem is we don’t have any conditions under which
that cell cares. But we’ll come back and even
do more because we could do things here. So now what are we
going to do? How are we going to see where
our beta globin is? AUDIENCE: [INAUDIBLE]. ERIC LANDER: Make the protein. So we’re going to
persuade this E. coli to make the protein. And how are we going to
recognize the protein? AUDIENCE: [INAUDIBLE]. ERIC LANDER: So how can we
recognize a protein? AUDIENCE: Function. ERIC LANDER: Sorry? AUDIENCE: Function. ERIC LANDER: Function. The problem is we didn’t really
have a function to complement. Electrophoresis. We could actually take every
colony on the plate, grow it up, purify the proteins, and
see if there was a protein band at just the right place
for beta globin. We’d have to look at about a
million colonies to do it. Maybe a little more, actually
more because you know, statistics and all, 10
million colonies. And the issue there is graduate
students tend to, you can check with the TAs, but on
the whole, tend to rebel at the thought of prepping protein
from 10 million separate cells. How else can we tell if our
cells are making beta globin? AUDIENCE: You add
a marker to it? ERIC LANDER: Something that
sticks to beta globin. Turns out that antibodies are
really good for this. If you take beta globin and you
inject a mouse with beta globin, it’ll make an immune
reaction against it. And we’ll learn more about the
immune system in the course, but it will actually make
antibodies that bind to beta globin. So suppose I told you there were
antibodies that actually bind to beta globin? Now, what can I do? AUDIENCE: [INAUDIBLE]. ERIC LANDER: I’ll make it a
fluorescent antibody, sure. You can have a fluorescent
antibody if you want. Fluorescent antibody
that knows how to bind to beta globin. Now, how do I find my cell? AUDIENCE: Colonies. ERIC LANDER: Take my colonies. Do what to them? AUDIENCE: Add antibodies. And look for the colony
that had antibodies– ERIC LANDER: Stuck to it. I just wash antibodies
over my colonies and see where it sticks. Now, the only details are this
is on an agar plate, and it’s really messy to do that. So just technical details of
pulling that off are I actually grew these on a little
piece of filter paper. So I put a piece of
filter paper down. I grow the colonies on filter
paper getting their little nutrients through there. I take off my piece of filter
paper that has my colonies growing on it. I need to crack open all of
those cells, and it turns out I can do that by a chemical
treatment that will crack open all the cells and leave
their protein contents just spewed out. They spew out their guts
right here, each cell. And then I wash my antibody
over it, and my fluorescently-labeled antibody
sticks to that guy telling me that beta globin was
right there. Any questions? I did it. You did it. Except there’s a problem. What was your problem? AUDIENCE: Is it possible that
the antibody won’t stick? ERIC LANDER: It’s a wonderful
antibody. It’s a perfectly specific
antibody. The antibody is totally
perfect. It only sticks to beta globin. Does the bacteria want
to make beta globin? You’re throwing this stuff in
and if any bacteria actually did make you beta globin, this
would be working perfectly. The little problem is, will a
piece of human DNA thrown into E. coli make beta globin? No. Why not? Well, let’s go back to
our understanding of how genes work. The beta globin gene is a
locus that has a human promoter which then goes
and makes a transcript. That transcript has
multiple exons. Those exons go into the
transcript and are spliced together to make a
mature message. Here is the mature message,
which gets translated. We have splicing, and then
this gets translated. The spliced product
gets translated. And what’s the problem asking
E. coli to do this? AUDIENCE: It doesn’t
do splicing. ERIC LANDER: It doesn’t
do splicing. And in fact, if you told me
we’ll use yeast instead, it turns out yeast does a very
bad job in splicing human introns also. And E. coli doesn’t recognize
human promoters. So this doesn’t work
very well. Any takers on what we’re
going to do about this? How do we fix this problem? AUDIENCE: [INAUDIBLE]. ERIC LANDER: Ah. So one solution would be teach
E. coli how to splice DNA. That’s hard. Start with cDNA. That’s nice. So let’s take our messenger
RNA, mRNA, and let’s now convert it into cDNA. What is cDNA? cDNA remember, we can
copy RNA into DNA. What enzyme copies
RNA into DNA? We learned about it
when we studied replication and all that. AUDIENCE: Reverse
transcriptase. ERIC LANDER: Reverse
transcriptase. So if I can get my hands on some
reverse transcriptase, I would be able to add reverse
transcriptase to mRNAs from a human cell and make cDNA. And where do I get reversed
transcriptase? It’s in the catalog. So I now make cDNA and
instead, I put the cDNA in the vector. I do this, of course,
for many, many, many different RNAs. I take total RNA from the human
cell, I convert all the total RNA all at once into
different cDNAs. Those different cDNAs get put
into the vector, and I now have what’s called
a cDNA library. So now I have a cDNA library. And now each of these guys has
not a piece of human genomic DNA, but a piece of human cDNA
copied back from a message. One of those guys
has beta globin. Now will E. coli produce
beta globin? No. Because AUDIENCE: Promoter. ERIC LANDER: Promoter. It doesn’t have a promoter
that it recognizes. What are we going to do
we do about that? AUDIENCE: Add one
in the vector. ERIC LANDER: Add one
in the vector. Give it a promoter
in the vector. Good. You guys are getting into
the engineering of this. Let’s say that the vector
has an E. coli promoter. Now, each of these cells will
actually make that mRNA. That mRNA is spliced,
and it will, in fact, produce an mRNA. The cell will translate it if
we do this right, and is the genetic code the same
in E. coli in human? Yup. So we can actually make, you
could make beta globin. Now, we wash over our antibody,
it recognizes which cell is making beta globin,
and it sticks. There’s beta globin. It works. Oh, by the way, what have we
also just invented here? We’ve invented a bacterial cell
that’s able to produce a human protein. What if instead of beta
globin, we put in the gene for insulin? What would we have invented? A way to produce insulin without
having to purify it from the pancreases of animals,
which is how we used to have to treat juvenile
diabetics. But instead, if we put the
insulin gene in, and we have an E. coli promoter, let’s
say, in theory, we can make us insulin. And what would we
have invented? The biotechnology industry. That’s basically the start to
the biotechnology industry. One of the first things that
was done was cloning the insulin gene to make
recombinantly-produced insulin by just the method you
guys invented today. It’s just unfortunately others
invented them first, so you don’t get a patent on it or
anything, but all right. So now we got two ways to clone
genes so far, and there are a lot of ways
to clone genes. I’ve given you two, later
in the course, I’ll give you a third.

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  1. Good lecture – but read Craig Venter's autobiography and there's some pretty shocking behaviour reported there from Dr Lander. 

  2. I have to write an essay on this, and was unsure of the steps. Thanks for the clarification, i wish my lecturers were this good at explaining! 

  3. @ 7:14 – 7:32
    One question, I suppose we're growing Beta Globin cells for the purpose of using it?
    If the antibody sticks on the Beta Globin cells, these cells become un-usable?

    Anyone who knows the answer, pls share your knowledge.

  4. Oh my god, I wish I had a teacher like this! Perfectly understandable AND funny? It doesnt feel like studying anymore 😀

  5. Learning in the United States seems so easy. In India, in the same amount of time, you need to process five times more data due to the competition and resulting higher floor of knowledge.

  6. sun everything comes from it, photosynthesis, next next. and d 4 terrestrial plan will take when suns color is our red.

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