Basic Mechanisms of Cloning, excerpt 1 | MIT 7.01SC Fundamentals of Biology

Basic Mechanisms of Cloning, excerpt 1 | MIT 7.01SC Fundamentals of Biology

Articles, Blog , , , , , , , , 35 Comments


PROFESSOR: So, first step,
we need to cut our DNA. Step one, cut, which is going to
be DNA restriction enzymes. It turns out, quite remarkably,
that if I have a sequence of DNA, five prime A G
C T A G A A T T C T T A C C three prime, and we’ll
come backwards filling in the sequence. It turns out that molecular
biologists are so cool that they invented a protein that is
able to recognize that six letter sequence, G-A-A-T-T-C. And it is able– actually it’s
G-A-A-T-T-C on this strand, but what is it coming back? It’s G-A-A-T-T-C. It’s
the same thing. So actually this is
a palindrome. That’s kind of nice. It’s a palindrome– it’s a reverse palindrome. It’s the same word spelled
backwards on the other strand. So what it does is it
cuts it like that. And what it produces is a DNA
molecule like this and a DNA molecule like that, that’s
mostly double stranded, but has a four base pair overhang. The overhang reads
T-T-A-A here. It reads A-A-T-T there. And remember this is five prime
to three prime, five prime to three prime. And there you go. This guy has its little
phosphate at the end there. This guy has his little
hydroxyl over there. And it cuts it. Now that is an incredible
piece of engineering. To come up with a protein, to
devise a protein, that is able to recognize those six bases
and cut at those six bases. And cut in just this
way making a really clean overhang here. It’s this cool five
prime overhang. Who do you think invented
this cool protein? What engineer came up with
this cool protein? AUDIENCE: MIT engineers. PROFESSOR: MIT engineers,
yeah. Not a chance. Not a chance. This is a really tough feat. This is something that can only
be done by the smartest engineers on the planet. And MIT engineers are
unfortunately only the smartest human engineers
on the planet. Who came up with this
is E. coli. AUDIENCE: So you found it
somewhere in nature? PROFESSOR: Sorry? AUDIENCE: You found it
somewhere in nature? PROFESSOR: Of course you find
it somewhere in nature. Almost everything important
that we say molecular biologists have come up with, it
means molecular sat at the feet of the true masters,
bacteria, and learned from the true masters. This protein is found
in nature. And it’s found in E. coli. In fact, it’s found in E. coli
strain R. And it was the first such protein found in E.
coli strain R, so it gets the name EcoR1. And it cuts the DNA like this. Pretty cool. Pretty cool. Now, it turns out that E.
coli has this EcoR1. How often does E. coli– so whenever I take EcoR1, this
protein, purified from E. coli, and I add it to DNA it
always cuts at this site, which we call an EcoR1 site. How frequently do we
expect, what’s the frequency of EcoR1 sites? G-A-A-T-T-C, how often will
that occur at random? One in– AUDIENCE: Two to the sixth? PROFESSOR: One in two
to the sixth? How many letters do I have? AUDIENCE: Four, oh,
four to the sixth. PROFESSOR: One in four
to the sixth. My frequency should be about
one in four to the sixth, which is about what? What’s four to the sixth? It’s two to the 12th. It’s about 4,000. It’s about one in
4,000 letters. One in 4,000 bases. So it’s very convenient. One in every 4,000 bases
it’ll roughly cut. It’ll cut at roughly
one 4,000 bases. Why doesn’t E. coli
cut its own DNA? If it’s got this protein
floating around in its cell, why isn’t it chopping
up its own DNA? Doesn’t have G-A-A-T-T-C? Yeah, the problem is
it’s so frequent. That’d be really hard
to make sure– I mean, E. coli has 4 million
letters in its genome. This should cut every
4,000 bases. You expect about 1,000
such sequences. It might be hard to arrange
not to cut– not to have any such
sequences. It’s a good idea, is not to have
any, but an alternative– AUDIENCE: [INAUDIBLE]. PROFESSOR: It protects them. It turns out E. coli, instead
of avoiding the sequence altogether, has another
trick up its sleeve. E. coli protects this sequence
whenever it occurs. So it turns out that whenever
you have a stretch of the E. coli genome that has this
G-A-A-T-T-C in it, what E. coli does is it puts– I’m just writing M-E here
for a methyl group. Right, C-H three up there. It puts a methyl group– I’ll write C-H three. There we go. It puts a methyl group on
the A, that middle A. Well, that is a cute trick that
E. coli uses, putting a methyl group there. Because what happens is, when
there’s a methyl group, right at that position, the enzyme
no longer recognizes and no longer cuts there. So that’s kind of clever. E. coli makes this protein
that can recognize G-A-A-T-T-C, but it has a
second protein that puts methyl groups there. And this protein happens
by accident to be called a methylase. It has a methylase. And the methylase protects
that sequence. So now, this is really cool
engineering, but kind of dumb. What’s it doing there? It has something that cuts the
sequence and it protects the sequence, why bother
having this? Yeah? AUDIENCE: You can use it to cut
it at places to unwrap the true strands. PROFESSOR: That’s an
interesting idea. We could use it to cut our DNA
and open it up to unravel our true strands. It’s a thought. Yes? AUDIENCE: To protect the
bacteria from viruses? PROFESSOR: Protect the bacteria
from viruses. How do you protect yourself
from viruses? Well, you have an immune system
with immune cells and antibodies and all that. Does E. coli have an
immune system? Why doesn’t it have
immune cells? Because it’s like one cell. How’s it going to have an
immune system, right? So suppose E. coli
gets a cold. Suppose it gets infected
by a virus. How’s it going to
protect itself? Cut at a frequently occurring
DNA sequence. Now the virus, of course, isn’t
methylated there, bingo. That’s how it tells its own– you can tell cell
from an invader. E. coli can tell cell from
an invader because it’s methylated its own G-A-A-T-T-C
sites, but the virus isn’t methylated there. Way cool. This is an immune system
for E. coli. Now, it turns out– so
this is protection. These restriction enzymes
protect E. coli from viruses. It turns out that E.
coli is not alone in this clever trick. It turns out that other bacteria
have also thought of this trick. So it turns out that there is
another restriction enzyme that cuts at G-G-A-T-C-C. And
on the other strand it goes G-G-A-T-C-C. It, again, cuts in
that distinctive pattern. And it’s called BamH1. And there’s another guy. And he cuts at A-A-G-C-T-T,
A-A-G-C-T-T. And it also cuts like that. And it’s called HindIII. And there’s some that
cut at G-A-T-C, just the four letter word. And they cut like that. And there’s some that cut at
C-A-G-C-T-G, C-A-G-C-T-G, and this, cuts smack
in the middle. In other words, there’s a wide
number of different tricks. Some cut at six bases. Some cut at four bases. Some cut at eight bases. Some cut leaving an overhang. Some cut smack in the middle. Some cut leaving the overhang
in the other direction. Some allow a degenerate
base in the middle. It doesn’t care which base
is in the middle. There’s a zillion different
solutions that bacteria have come up with for their
immune system. And so, if I want to cut up some
human DNA all I need is say, this protein EcoR1 or BamH1
or HindIII or MVL 1 or PVU 2 or et cetera. And I can do that by growing
up E. coli and purifying the protein. And if I wanted HindIII, I
would grow up haemophilus influenza and purify
the protein. So in a molecular biology lab,
today, if you want to cut up human DNA, you could grow up
some E. coli and purify EcoR1 or haemophilus influenza. And that is indeed what ancient
molecular biologists did in prehistoric days in
the 1970s and 1980s. They would purify their own
restriction enzymes. They’re still alive today. You can talk to them. There are many of them
on the faculty. And they’ll tell you how it put
hair on their chest to be able to purify their own
restriction enzymes. What do you do today? Order it online from
the catalog, right? You know, there’s the
catalog, the New England Bio Labs catalog. Let’s see what we got here. Restriction enzymes, modifying
enzymes, polymerases, all right, EcoR1, sale on
EcoR1 right now. $100 buys you 10,000
units of EcoR1. It’s in the catalog. You can go online. You can order it. You can have it tomorrow
by FedEx. So, but that’s how it works. It’s in the catalog. So you can get any restriction
enzyme you want to cut DNA anywhere you want to.

35 thoughts on “Basic Mechanisms of Cloning, excerpt 1 | MIT 7.01SC Fundamentals of Biology

  • Peruviantank Post author

    So do these pair always like come together or are meant to be like destiny if you find your soul mate. (A) always matches with (T) as well as (C) matches with (G). Question isn't there like a special case or one of the those rules that the letter (C) has to be paired up with (A) but what about (T). C=A/T I don't know πŸ˜‰ just guessing and I took a stab at it and I'm pretty sure it doesn't make sense but since I'm coming off a math mind since all I learned in my classes in OSU is manipulation.

  • Peruviantank Post author

    Plus I do apologize with all of these series of question but I'm a student who is kind of curious to learn pretty much that sounds like a challenge and have something to do with science or mathematical formulas. πŸ™‚ Lo siento. But hey I rather be doing this then writing a book. Playing with letters and numbers don't sound difficult. It's child's play to me once I understand why.

  • Peruviantank Post author

    So basically what Eric Lander did was once he found out the first ladder I assume then flip the second and reverse it. I'm not so sure if its true. I'm just guessing here.

  • Peruviantank Post author

    It's only pretty cool if we can experiment mutiple of times and apply the formulas to the test lab.

  • Peruviantank Post author

    So the 4 he got is from AATT or TTAA then the 6th power he got from the letters then 4 to the 6 is 20000 is number of possiblities of where it can land.

  • Hafizah Hoshni Post author

    16 Basic Mechanisms of Cloning, Excerpt 1 (00:13:19)

    Thanks !

  • Kevin Rodriguez Post author

    The four he got was from the possibilities of bases (A,T,C,G) that can occur at one site. The six comes from the number of spaces he is trying to fill. (GAATTCC)-> (_ _ _ _ _ _) .

  • john asd Post author

    You are an excellent lecturer. Hope your students understand your great value.ο»Ώ

  • Ammar Ahmedani Post author

    Why not bacteria do the same with acquired gene e.g. gene of resistance acquired fro other bacerium ? Is it cleaver to the extend?

  • Zheng Chao Bong Post author

    Subscribed =)

  • Rosa Costa Post author

    I wish this lecturer was my teacher, he is very skilled

  • BΓ­ch Ngọc Cao TrαΊ§n Post author

    I love professor Eric Lander!

  • Shellstorm1 Post author

    Great for the mind of a bored 12 year old

  • Ta-Ta Huang Post author

    Best introductory lecture ever

  • Veera Selvan Post author

    Very excellent teaching l like that Eric Lander sir very great !

  • argyrophilic Post author

    Update: Ecor1 in 2018 costs $60 for 10,000 units. This is $40 less than the 2012 price of $100 for 10,000 units.

  • kundhanathan biology Post author

    bingo

  • Fluxpistol Post author

    lol basic

  • kundhanathan biology Post author

    oooooooo it protect bacteria from viruses….i am impressed

  • Mohammed A.modu Post author

    waw! so, interesting . Really , your are a great scientist

  • Vicky OH Post author

    I really like this professor Lander.

    But, who got the CRISPR-Cas9?
    ** drops microphone **

  • Sura T Post author

    Excellent πŸ‘Œ!! Great lecturer!! Engaging, passionate and personable. Those MIT students are so lucky to have a scientist like this as their professor!! None of that unfounded arrogance you see from other professors who are not half as good as this man.

  • juliana ocampo Post author

    I love the energy he has makes me want to learn more and more and more ! #biomajor. The 21 people that disliked this , need to find a new major.

  • Zheng Xu Post author

    molecular biology is boring. But This class is really interesting. well done

  • Fiza Farooq Post author

    Those who disliked his video are seriously dumb.why people don't appreciate a skilled person.

  • Ayisha Mohammed Saliya Post author

    β€οΈπŸ‘πŸ»

  • p-h chan Post author

    Amazing teacher!

  • p-h chan Post author

    Cleared many of my frustrations in this topic.

  • Raki's Just Bio Post author

    Just loved your way of explanations sir. Hats off to you.

  • Brainstorming Plus Post author

    πŸ‘πŸ‘πŸ‘πŸ‘πŸ‘

  • ι‡Žη£ε…ˆθΌ©γ‚’η ”η©Άγ™γ‚‹ε­¦θ€… Post author

    εˆΆι™ι…΅η΄ γ γ€€ζ‡γ‹γ—γ„

  • Anil Kumar Sharma Post author

    genome mixing of all types of oil plants together will gives a complex compound for fractional distillations and patrol solution become easy forever

  • Anil Kumar Sharma Post author

    found that genome which gives oils in plants so we got directly patrol from trees and oil plants genome mixing

  • Brainstorming Plus Post author

    πŸ‘πŸ‘πŸ‘πŸ‘πŸ‘

  • Anthony Kernich Post author

    this dude explained a whole lecture series in 10 minutes

  • Anil Kumar Sharma Post author

    gives number to the genome and we found that limit and structure and size and arrangement of any genome
    probability gives total number of combination and permutations and pauli exclusivity principles the number of elements repetition and we got the ultimate behaviour of genome mixing gives desired resultπŸ˜‹πŸ˜‹πŸ˜πŸ˜πŸ˜

Leave a Reply

Your email address will not be published. Required fields are marked *