[ Silence ]
>> Okay, welcome back.
Quick sound check.
Everything okay?
Great, thank you.
Welcome back.
Today, we're going to be
finishing up the topic
that we were talking
about last time.
Last time, we were talking
about combinatorial approaches
in chemistry and then we'll
talk a little bit more
about combinatorial
approaches in biology.
And I'll show you a
couple of examples of this.
All right.
Okay, that's interesting.
All right.
Okay, so again we're here.
We just completed our
survey of biomolecules.
I'm going to complete the
topic of making combinations
of biomolecules and
then we'll talk
about tools for chemical
biology.
And this is really important
because these are the tools
that you're going to be using
when you write your proposals.
So I'm glad you're
all here today
because you absolutely need
to hear this to be able
to write a good chemical biology
proposal which recall last time,
I told you was going
to substitute
for the final exam
in this class.
There is no final
exam in this class.
We will not have a final.
Instead on the very
last day of class,
you will hand me a 10-page or
so proposal, a written proposal
with figures and it'll
be an original idea,
something that no one in the
planet has thought of before.
You will be the first.
And it's going to be really
fun because it's really great
to come up with creative ideas
and that's really the
ultimate goal of science.
Science is really a
creative enterprise.
Our goals are to
invent new concepts,
to tell people new visions of
the universe and to do this;
we have to somehow invent
these new experiments to do.
Okay, so I'm going to be talking
to you today about the tools
in your toolkit that
you're going to be using
to do this assignment.
Okay, I already talked
about these announcements.
I'm skipping some stuff.
Oh, office hours.
I had office hours
yesterday that got derailed
by a student emergency
and I know at least one
of you sent me an
email about that.
I apologize.
I will have office hours
today and in addition,
I sent an email back
to that student.
So I apologize if you
came by yesterday.
There was a student
health emergency
that absolutely needed
my attention and so I had
to close my door
to deal with that.
Okay, so apologies there.
Other office hours, tomorrow,
Mariam will have her office hour
on Friday and I'm hoping
Kritika will be back next week
and I'll introduce you to her
and she'll have office
hours next Tuesday.
Okay, so all right,
any questions about any
of the announcements,
things like that,
things that we talked
about last time?
Questions about the
course structure?
Oh, I got an email from someone
and I apologize for
not replying.
The email was, "When are you
going to post online the slides
that I'm flicking through?"
And the answer is I'm going
to try to get to that today.
And then my plan is to
basically post all of my slides
from the previous
year and so that way,
then at least you'll
have a guideline
for what the slides
will look like.
Chances are, I'll
heavily modify these
or slightly modify
these depending
on how much time I have
before each lecture.
I mean, literally five
minutes before the lecture,
I was making changes
to the slides.
It's almost impossible to
stop me from doing that.
I just love this too much.
So because of that, I'll be
posting kind of a guideline
for what the slides will
look like in advance.
And then I'll come back
with something that's
more definitive.
Okay, so at the end of today's
lecture, then I'll post all
of the week one slides
in a definitive way
but I'm also going to
post last year's week two,
week three, week
four, et cetera.
Okay, sound good?
Okay, any questions about that?
Okay, great.
Okay so let me review what
we talked about last time.
If there are no questions about
any announcements or things
like that, we're going to go
straight into the material.
Okay, good.
So what we talked about
last time was the definition
of chemical biology.
Chemical biology uses
techniques from chemistry,
often new techniques from
chemistry, often techniques
that had been invented
specifically to answer problems
of biology but not always.
And then these techniques
from chemistry are used
to address understanding
biological systems
at the level of atoms and bonds.
That's the goal of
chemical biology,
to really understand how
organisms are living,
how they do the things they do
at the level of atoms and bonds.
Okay, so I'm really
fascinated to know
about that hydroxide
functional grid
that donates a key hydrogen bond
or provides a key Bronsted acid
to some mechanism in
an enzyme-active site.
That's the part that
makes run to work,
the sort of the details of this.
I basically want to use the
arrow pushing that you learned
in sophomore organic
chemistry to explain biology
and that's the goal
of this class
and that's the definition
of chemical biology.
So last time, we learned
about two key principles
that organized biology.
The first of these is essential
dogma which provides the roadmap
for all biosynthesis taking
place inside the cell.
Everything that the cell
has to synthesize will flow
through this central dogma.
This is the flow of information
for biosynthesis by the cell.
So everything that your cells
will synthesize is going
to be encoded in some way by
the DNA inside your cells.
Oh, and can I ask you if you
have an empty seat next to you
to move over to the
right just to open
up some seats on the edges.
Some people I know are coming in
from other classes so you know,
so other classes that are ending
about when our class
is starting.
So if you have an empty
seat on your right,
if you can just scooch over
and leave seats on the edge,
that would be really
appreciated.
Okay, thank you.
Okay, so the second
key principle
that we discussed was evolution.
Evolution provides a principle
that helps us organize vast
amounts of knowledge and really
in the end simplifies
biology enormously.
And it's actually a principle
that all of you are going
to be applying when you
design your chemical
biology experiments.
Because I will tell
you in advance
that I will not accept
any proposals
that involve experiments
on humans, okay?
So experimenting on humans
has its own special topic
that I can actually
teach a whole quarter on.
Okay, it requires
ethical considerations.
It requires tremendous
design considerations.
It's not nontrivial to
sample, for example,
a diverse population
of humans and ensure
that you're getting diversity.
So all of those considerations
are beyond the realm
of this class.
So instead, what I'm going to
ask you to do is experiment
on non-human organisms.
You might for example
choose cells from humans
or you might choose
model organisms.
And by choosing those
model organisms,
you're applying a key principle
from evolution which is
that that model organism
descended
from some common ancestor
that we share and in doing so,
acquired the same mechanisms
that govern its chemistry
and its chemical biology.
And so that means,
if we learn something
about this model organism, we
can then apply that knowledge
to understanding
how humans work.
Now naturally, there's
limits to this, right?
If your model organism is a
salamander and you're interested
in understanding how the
salamander regenerates its arms
when you cut them off,
which incidentally would be an
absolutely fascinating topic
for a proposal, right?
There's a limit to how much
analogy you can do back
to humans, right?
We humans don't have that
same mechanism obviously
and it would be absolutely
fascinating for me to learn
from you how it is that you
plan to apply the biochemistry
that you are learning
about stem cell growth
to develop say limb
regeneration in humans.
I would love to learn that.
Okay, so evolution is important
to us because it tells us
that fundamental processes
are more or less the same
for every organism
on the planet.
And I'll be showing you a few
examples in the next few weeks
that illustrate this
universality
of chemical mechanisms.
In addition, we also saw that
evolution is really a tool
by which we can evolve
molecules to do powerful stuff
for us inside the
laboratory and I want to pick
that topic up for us today.
Okay, so I'm going
to start there.
Any questions about anything
that we saw on Tuesday?
Okay, now I also got some
really fascinating emails
from some virologist in
the audience who pointed
out there's actually
the coronavirus protein
that is known to start
with an RNA template
and then replicate RNA and
that's absolutely fascinating.
I wasn't aware of that.
So there are exceptions
to what I'm teaching you.
I'm going to try to teach you
the sort of most general thing
and yes, there will
be exceptions.
Don't hesitate to
point them out to me.
I'm fascinated by
those exceptions too.
Okay, so let's pick up where --
okay, before we do, last thought
about this proposal assignment.
To do the proposal successfully,
what you have to do is you have
to come up with a
novel idea, okay.
I will not accept any proposals
that don't have something
new in them, okay?
And I will actually ask the
TA's to do Google searches
and literature searches in
PubMed and other sources
to verify that what
you're proposing
to do has not been
done before, okay?
So you have to come up
with a creative new idea.
This sounds daunting but let
me provide some guidelines
on how to do this, okay.
So the first thing that
you need are a series
of experimental tools and
then knowledge of the problem.
Okay, so experimental tools, I'm
going to provide to you today.
I'm going to give you a
toolkit by which you can go out
and start to address
problems in chemical biology.
The second portion,
knowledge of the problem.
You need to know that actually,
you know, there's a key step
in limb regeneration that's
not so well understood.
That second step comes from
reading the literature, okay?
And the first assignment
in this class,
the journal article
report is designed
to help you address
this second thing,
knowledge of problems, okay.
So in doing the assignments
that are required for the class,
these two things are going
to come together, okay.
Today, we're going
to address number one
and then item number two, you're
going to get by Valentine's Day,
February 14th, you'll have a
journal article report and then
in doing this assignment, you'll
be looking at the literature
and you'll start to identify
problems in the field
that interests you, okay.
So you'll choose a journal
article that's relevant
to your interest.
I don't know what
your interests are.
Let's say you want to be
a dermatologist, okay.
Maybe you'll find a
chemical biology report
that uses skin cells and looks
at say melanoma development
in skin cells and looks at it
at the level of atoms and bonds.
I would love to hear
more about that.
And then by doing this
assignment, you'll start
to know what are
the big unknowns
in skin cell tumor
development, okay?
What are the things that people
are fascinated by that are --
they're designing
experiments to address.
And you'll have the tools from
this lecture that will allow you
to address those problems.
Okay, sound good?
Okay, so how to find
the problem.
The first thing I
need to ask you
to do is start reading either
Science or Nature, okay?
So I assume many of
you are science majors.
If you're not a science
major, raise your hand.
Okay, you're a fascinating case.
I'd like to talk to you later.
So come to my office and
just introduce yourself.
Okay, so everyone else
is a science major.
You're going to get
a degree in science.
I'd like you to read either
Science or Nature pretty much
for the rest of your life.
Pick one. You don't have to
read a book and furthermore,
you don't have to read
them all that carefully.
Just skim through them.
By doing that, you will be
an informed citizen, okay?
You will know more about
Science than 99.99%
of the people in this planet.
And furthermore,
you'll learn something
about what's really
cutting edge, okay?
You only have to spend 10 or 15
minutes flipping through Science
or Nature, just looking at
the headlines and saying, "Oh,
they discovered a new
class of quasars out in,
you know, some other galaxy."
Just doing that is
enough to help you --
well, it will certainly
have much better banter
at cocktail parties,
let's say, [laughter].
And to me, that's enough.
Okay, so this is part
of your education.
So start reading
Science or Nature.
Simply flip through them.
That helps you identify
problems.
The second way is to
look at PubMed or Medline
which are the same things
and I'll be talking some more
about PubMed in a
future lecture, okay.
So hopefully, you already
know what PubMed is.
Hopefully you already
know how to apply it.
I'll be showing you
how to apply it
to chemical biology
problems at a future lecture.
But these are the two ways that
you shift through literature
to find stuff that's interesting
and that grabs your attention
because in the end, you
want your proposal to be
about something that
really interests you, okay.
You're going to spend
a lot of time on this.
Okay, many, many hours
and if it's not something
that totally interests you,
that's not somehow related
to the bigger picture of
your career aspirations,
it's not going to
be as much fun.
And in the end, if it's
fun, you'll do a better job.
I'll get a better
proposal back out of it
and that's the part
that interests me.
Okay, now I was reading -- I
chair the Admissions Committee
in the Department of
Chemistry at UC Irvine
and I was reading the
application essays
from all the wonderful
applicants who have applied
to UC Irvine this year and I
came across this wonderful quote
up here, "The more you know,
the more questions you can ask."
And so those questions that you
can ask, those are the questions
that you will be addressing
with your proposals.
So our goal is to get your
knowledge up to the point
where you can start asking
those questions, okay?
All right, now I know
this is all very --
this all seems very abstract
but it's not going to be
as abstract in a moment, okay?
Sound good?
Questions so far?
All right, don't be too
daunted by the assignment.
It will all come together
when you're ready.
Okay, last announcement,
next week's plan.
Next week, we're going to
be starting on Chapter 2.
Please skim Chapter
2 in advance.
Take a look through Chapter
2 even before I get to it.
Chapter is the review
of arrow pushing.
Chapter 1 was a review of
the biology you need to know
and next week we'll be
talking about arrow pushing
and mechanistic organic
chemistry that you need to know
to do chemical biology.
Okay so next week, we're
going to have two lectures
on mechanistic arrow pushing.
Now, here's the deal.
I'll be out of town on Tuesday.
But I prerecorded
Tuesday's lecture [laughter].
And so I'm trying a little
experiment this year.
I understand that the video
from Tuesday's lecture,
the last Tuesday's lecture is
already available and is going
to be shortly posted
online, okay.
So I will send you the link
to last Tuesday's lecture
and at the same time,
I'll send you the link
to the next Tuesday's
lecture, okay?
And so that next Tuesday's
lecture then, you can watch it
in your pajamas, in the
comfort of your dorm room, okay?
And so we're going to try
that for Tuesday's lecture.
I think that's actual --
I think that will work
but I'll know very quickly
if it doesn't work, okay.
And then Thursday, I'll be back.
So Tuesday, I'll be at Cal
State LA giving a seminar.
Thursday though, I'll be back.
Okay, sound good?
Okay. All right, so that's
the next week's plan.
We're going to be
reviewing important stuff
from organic chemistry.
Mainly this focus is
on structure reactivity
of carbonyls.
If you were weak in 51C,
please reread this chapter
on carbonyl reactivity
structure and things like that.
There might be two or three
chapters for you to read.
Mechanisms involving carbonyls
especially the aldol reaction.
90% of carbon-carbon bonds
and chemical biology are
made using an aldol reaction.
You need to know what an
aldol reaction is, okay?
If this word "aldol" is totally
unfamiliar to you then you need
to spend a little bit of time
this weekend reading about it
and getting familiar
with it again, okay.
Because I'm going to assume that
you know about an aldol reaction
when we get to it, okay?
Now, on the other
hand, in your review
of sophomore organic
chemistry, don't get worked
up about reactions
where the synthesis
of carbonyl-containing
compounds.
Anything that you
learned in 51C about how
to make the carbonyl
using PCC is more
or less worthless
for this class, okay.
Because PCC is not
found in cells.
It's totally toxic
and so good news.
As you're skimming through -- as
you're reviewing, if necessary,
don't get too worked up
about memorizing a bunch
of name reactions and
stuff like that, okay?
Instead focus it on mechanisms.
Focus on the reactivity.
Understand how carbonyls
work, that sort of thing.
That's what you really
need to know going
into the next few
weeks of this class.
Okay, that was a long
set of announcements
but thanks everyone for
coming out for that.
All right, let's get started on
the actual -- the new material.
I want to talk to you today
about combinatorial
approaches first.
And I'm going to pick
up on the last slide
that I showed you last time and
make sure that I didn't skim
through it so quickly that it
didn't make any sense to you.
And then we'll go on
to the next topic.
Okay, so last time,
oops, I was talking
about modular architecture
in organic synthesis.
This is a -- whoops,
that's not what I wanted.
Just give me one moment
to figure this out.
All right, I guess we'll
have to live with this, okay.
So modular architecture is a
design principle that allows you
to synthesize compounds in
a way that allows access
to combinatorial libraries.
And last time, we talked
about this principle
of combinatorial libraries.
Combinatorial libraries
are big collections
of different molecules and
in a combinatorial library,
you have a different set of
modules that are shuffled around
and recombined in a way
that makes a whole series
of different molecules, okay?
And we talked last
time about this class
of compounds called
benzodiazepine.
This name should be --
the name of this class
of compound should be
vaguely familiar to you.
this is an important class
of compounds that's
found almost ubiquitously
in medicinal chemistry
and they're used
for amongst other
things, antidepressants.
So you could make a
combinatorial library based upon
this benzodiazepine scaffold
by varying the R
functionality shown here.
And you do this by a very
straightforward synthetic plan
that involves the recombination
of a ketone together
with an aniline so
this is a compound
that has both the ketone and an
aniline functionality together
with some sort of alkyl
halide and an acid,
let's just say an acid
halide and an amine.
And so these will
all snap together
to give you this
benzodiazepine framework.
I'm not showing you the
mechanism for this and it's not
so important for our discussion
so we're going to skip over it.
But you can imagine having say,
you know, 20 different versions
of this ketone-based
compound with different R1's
and different R2's, 20 R3's
over here or 20 compounds
that have different R3's
and then say, 25 compounds
that have different R4's.
When you put these all
together and you would do this
in individual reaction flasks,
you'll end up with a large
number of different compounds.
Okay so let's just
do 20, 20, 20.
Okay so 20 of these, 20
of these, 20 of these.
If we make all possible
combinations of those,
how many compounds
will we end up with?
How many benzodiazepines?
20 times 20 times 20.
>> So third power.
>> 20 to the --
>> Third power.
>> Third, which is --
>> 8000?
>> 8000. Thank you.
Okay, you guys are
scaring me now [laughter].
Okay so 8000 compounds can
very readily be synthesized
by starting with simply 60
different precursor compounds.
And that's pretty powerful.
If you have 8000
different benzodiazepines,
each one that is potentially
some bioactivity then
that collection could have a lot
of very powerful new therapeutic
compounds in it, for example.
Okay and then we talked
about some other
different modular frameworks
that can be used.
Now, I want to shift gears.
That's an example of using
combinatorial chemistry
in the synthetic laboratory.
This principle, of course,
borrows heavily from biology
and it turns out that your
immune system uses a similar
principle to develop diverse
molecules called antibodies
which are one of the
first lines of defense
against foreign invaders.
Okay, so if heaven forbid,
you decided to take the apple
off the ground over there
and start chewing away on
it, you would find a lot
of foreign bacteria
in that apple.
And so likely antibodies
would play some role
in fighting off those
foreign bacteria.
Okay, so here's the
way this works.
So antibodies' job is
to be binding proteins.
Their job is to grab on
to non-self molecules.
So I'm going to refer to
this class of compounds
as professional binding
proteins.
That's what they do
for a living, okay?
That's their profession.
And it's one of the immune
system's first lines of defense.
Structurally, they
look like this.
I told you earlier, one
convention for looking
at protein structures
using a ribbon
to trace out the backbone.
I didn't tell you really
what these arrows mean
and these curlicues.
We'll get to that later.
But a different convention
for looking
at protein structures just maps
the surface onto the outside
of the protein structure.
Okay, so if you were
able to have, you know,
special electron
microscopy eyes, you know,
eyes that had amazing power of
resolution and vision ability,
what the antibodies
really would look
like is something like this.
Okay, so they have this
sort of bumpy exterior.
Now, the stuff down and
I've colored this antibody
to highlight its
structural components, okay?
So antibodies, it
turns out are composed
of a total of four chains.
Two of these chains are
called light chains.
They're shown here at the top
in green and then they're sort
of cyan color and
this purple color.
And then there's
two heavy chains.
Okay, the detail is
not so important.
Don't get worked up about
memorizing how many chains each
protein has.
Here's what's important.
Okay, antibodies have evolved
a mechanism that allows them
to recognize diverse
binding partners.
And they do this by having
a series of flexible loops
that can accommodate
different shapes
that they need to bind to.
Okay, so I'm turning now to
the very tips, the tippy-top
of the antibody appear which
is labeled binding site.
This is where the antibody
will try to attempt to bind
to that foreign invader.
Let's say you picked up a virus
when you bit into the apple,
now the virus is floating
around your bloodstream.
So the antibody is going to
attempt to bind to the exterior
of this virus and if
we zoom in over here,
this is the tippy-top.
This is just the -- this
is called the FAB region
of the antibody so the
FAB region of the antibody
over here and you could see.
And then in this van
der Waals sphere,
this is an antibody
binding to a small molecule.
So it's binding to some target.
The exact target not
so important for us
but notice how the target
is cradled in these loops.
Okay, the loops are gripping
this antibody very gently
but oh sorry, they're
gripping this antigen gently
but the antigen is wholly
buried in these loops.
So these loops are flexible
to accommodate many different
potential binding partners.
That flexibility is critical.
That means they can recognize,
you know, virus one or virus two
or if you go to Ethiopia
and pick
up some totally different
virus, they will also pick
that one up too, you hope.
And at the same time, these
provide enough other types
of molecular recognition
which we'll talk about later
that allows strong
enough binding
to muster an immune response
and then the antibodies
basically sound the alarm.
The red coats are coming and
get the immune response to go
into high gear to start killing
off that foreign invader.
Okay, so very first
line of defense
against foreign invaders.
Now, the problem and
the big challenge is
that these antibodies
need to recognize stuff
that your human organism, you,
have never seen in
your life, okay?
That means that if you travel
to India or you travel to,
I don't know, Palos Verdes or
wherever it is that you travel
and you pick up some
new organism
or some new foreign
invader, the antibody,
the combinatorial library
of antibodies needs
to be ready to recognize that.
And of course, you know,
this stuff has never
been seen before.
The antibodies have
never trained on that.
So the antibody -- the strategy
that your immune system uses is
to have a vast collection of
potential binding partners.
Okay, so make a big collection
of different antibodies,
each one with structural
differences to be ready
to recognize any particular
type of invader, okay?
Now here's the other thing.
So the size of the
collection is huge, okay,
and these antibodies
are produced
by immune cells called
B cells which look
like this, or B lymphocytes.
This collection is
fairly enormous.
It's estimated to be on the
order of about 10 billion
or so different antibodies.
Okay, but earlier, I told you
that the human genome is
only about 24,000 genes.
Okay so obviously there can't be
10 billion different molecules
in the immune system each
encoded by its own gene.
So instead the strategy that the
immune system has evolved is a
strategy whereby different gene
segments are recombined in a way
that then produces a
combinatorial library
of different antibodies.
Okay, so let me show you.
So there are 40 of these
variable genes, V modules,
25 diversity modules,
six joining modules,
and they're shown here.
So here's the V genes, the
D and the J genes and then
by combinatorial gene assembly,
these are brought together
to encode the antibody
heavy chain gene, okay.
So that encodes the
heavy chain that I showed
on the previous slide.
Similarly, the light chains
are produced by another type
of combinatorial gene
assembly whereby one
of these V's is picked
out and et cetera,
and one of the D's is
picked out, et cetera.
Okay, so in doing this, you
can get a very vast library
of different antibodies.
Furthermore, the antibody
diversity pool is further
diversified by a series
of genetic manipulations
that includes variable
gene joining.
So when the genes are joined
together, they're not sort
of glued together neatly.
Instead, there's little parts
that are clipped off or added in
and then furthermore, there's
a process called hypermutation
that goes through and
makes tiny little mutations
in the encoding sequences
as well.
So in the end, you end
up with around 10 billion
or so different antibodies,
each one different structurally
and potentially able
to recognize whatever
foreign invader you happen
to encounter during your life.
Okay, does it make sense?
Okay, so to summarize, what
we're seeing is a strategy
for combinatorial synthesis
that's used in the laboratory
and also used by your cells.
Okay, in both cases, there are
these modules that are shuffled
around and then rejoined
in literally random fashion
to give us a vast collection
of different molecules
and then we hope that these
different molecules are going
to be functional
when the time comes
that we actually need them.
Okay, make sense?
Okay, yeah, question over here.
>> For a C mutation, how do
[inaudible] because there's
so many of them and you know,
sometimes react then against us
because there's so many?
>> Okay, yes.
So there's a separate process
as it tracks out things
that recognize self as well.
>> Okay.
>> Yeah, that's an
interesting question as well.
So yeah, thanks for asking.
What is your name?
>> Joshua.
>> Joshua, okay.
Okay, changing gears.
So the last topic in Chapter
1 is a survey of the tools
that we need in chemical biology
to be able to address problems
and address the frontiers
of chemical biology.
So I'm going to have
a very quick survey
in the next 15 minutes or so.
I'm going to share with you
a series of different tools
that you can then use
in your proposals.
Okay, so think of
this as you're trying
to put together your toolkit.
This is going to be
the hammer, the saw,
the nail gun, whatever, okay?
So these are the things that
you need to put to address
to design experiments
in chemical biology.
Okay, so again, this is useful
for planning your
proposal assignments
but this also provides a
toolkit for further experiments.
We're going to be referring
to this toolkit quite
a bit in this class.
So later in the quarter, I'll
be able to say, "Oh yeah,
remember those antibodies
that I mentioned earlier?
Those are now going to
be in your toolkit."
This toolkit is very
diverse and vast.
It ranges from chemical reagents
to entire model organisms
and there's a huge
amount of diversity
in that range of
different tools.
So chemical biology as
a field uses all kinds
of different techniques.
It uses techniques
from molecular biology.
It uses techniques from the
very latest in nonlinear optics
and to image cells and
everything in between.
Okay, in addition, I also
want you to know these tools
because I want you to be able
to design experiments on the fly
to determine, you know, X. Okay
and a very common midterm
question for me would be,
"How would you design an
experiment to address, you know,
what kind of signaling,
chemical signaling is being used
by the gut bacteria,
your gut bacteria
to let their neighbors know
that sugar has arrived?"
Okay, which actually is a
pretty interesting question.
I'd like to know
how you'd do that.
Okay, in addition, I want you
to know how to describe negative
and positive controls.
We're going to be
talking about experiments
and all good experiments
have both negative
and positive controls.
So why don't we talk
about that topic first?
Okay, so if you're going to
be designing experiments,
you need to know first
what a negative control is
and what a positive control
is because you need to be able
to design these into
any experiment
that you want to design.
Okay, so good experiments
have both the positive
and a negative control.
Positive control first.
A positive control is a set
of experimental conditions
that provide an expected
response or a positive result.
Okay, so in this case,
you can basically want
to know does the conditions
in my flask produce, you know,
produce an amplified DNA
or something like that?
And so what you'll do is
you'll start with a sample
that you know should
work a certain way
in your experiment, okay.
It should give you a
predetermined result
and it should be completely
consistent every time.
It should be very --
it should give you
that expected result every time.
So this tells us that our
experimental apparatus is
working, okay.
And you need to know
this because oftentimes,
the experimental apparatus
in chemical biology labs isn't
simply a stirrer and you know,
a hot plate where you can
just test the hot plate
by sticking your fingers
on it for a nanosecond.
The chemical apparatus
might be, you know,
a tiny little microcentrifuge
tube and you've shot in a bunch
of different reagents.
You know, 10 different
reagents all of which are clear,
none of which you can really
assay all that readily.
So what you do is you set
up a set of conditions
where you know the
results and then you see
if the result is recapitulated
under your experimental
conditions.
Okay, so this is
a positive control
and you always want
to have one of these.
Good experiments have
positive controls.
Good experiments also
have negative controls.
So this is where you leave
out some experimental
condition in your experiment.
Maybe leave out the
test sample, okay?
So earlier, I was talking
to about trying to assay --
let's just say some sort
of microorganism found
in your stomach that responds
to the presence of sugar, okay.
And maybe you want
to know whether
that microorganism
releases indole to signal
to its neighbors, okay?
Actually that's not
a bad experiment.
So your experimental
apparatus will be measuring the
concentration of indole.
Your positive control
will be say some bacteria
that you know release indole
and that tells you whether
or not your experiment
is working.
The negative control can be
entirely missing the bacteria.
Okay, so you do the exact
same experiment but you leave
out the bacteria and no
indole should result.
Okay, if you see indole
resulting, that tells you
that you have a problem.
That tells you that you have
say, a contaminant for example.
This should result in a failed
experiment or a negative result.
So its experimental condition
missing a key element,
say the test sample, the thing
that you're trying to test.
Okay and again, it should
result in a failed experiment.
If it does not result
in a failed experiment,
that tells you that in your
conditions, you have some sort
of source of contamination.
You absolutely need these
negative controls, okay?
Because all too often in
chemical biology, we have lots
and lots of contaminants
and there are lots and lots
of false positives and we just
don't like that kind of thing.
You want to know that if you're
going to tell your friends
down the hall that you
discovered a new base
in the DNA sequence,
you want to know
that actually that's
the real thing, okay,
that you're not telling your
good friend something that turns
out to be totally wrong later
and it makes you look stupid
because no one likes
to look stupid, okay?
Now, because we have very
complicated experiments
in chemical biology that involve
lots and lots of variables,
remember I told you
earlier about the one
that has 10 different
things thrown
into little tiny
microcentrifuge tube,
we often have multiple
negative controls,
one for each possible variable.
Okay, so for example, you
might leave out the magnesium
from the buffer just to know
does the magnesium contribute
to this experimental result?
You know, is this actually
a magnesium-dependent enzyme
that produces indole
as expected?
If you leave out the magnesium
and you still are getting some
result that could tell you
that maybe it's not a
magnesium-dependent process.
Okay, so negative controls tell
you a lot about what's going
on in your experiments.
Okay and a good experiment
should have both negative
and positive controls.
Any questions about what
positive controls are,
what negative controls are?
Yeah.
>> So if you lined
up this thing,
if you failed positive control
and you passed the negative
control, do you [inaudible]?
>> Okay, this is
a great question.
It happens to me all the time.
Okay so the question is
-- what is your name?
>> B.
>> B? B, okay so B's question is
if your positive control fails
and your negative control works,
what does that tell you
about the experiment?
I would say that that tells you
that your experimental
conditions are worthless
and you cannot interpret
the experiment, okay.
Because if the positive
control fails
to work then you really
don't understand what's going
on in your experimental
condition, okay.
The positive control
really tells you whether
or not you understand
all of the elements
that compose your experiment.
If the negative control fails as
you expected it to fail, well,
maybe it's failing
because of the positive --
for the same reason that
the positive control failed.
Maybe you left out some
key reagent, right?
You know, maybe you didn't heat
it up to the right temperature
and hold it there for long
enough or something, okay?
So both your positive control
and your negative control have
to work in order for you
to interpret the results.
Okay, now I'm being
really dogmatic here.
I will tell you --
I will tell you
that we scientists
oftentimes look at experiments
that don't necessarily have
every control working, okay?
I'll look at those.
My students will show
me those all the time.
I'll look at them but I'm
not going to you know,
call up the Nobel Prize
Committee in Stockholm
and tell them about it, okay?
Because it's probably not worth
a lot of time but we'll use
that to guide the next
set of experiments.
We'll say, "Well what is it that
failed in the positive control?"
And then we'll design
and troubleshoot
and design the next experiment
using that information.
We'll look at the negative
control and say, "Oh yeah.
That failed.
That failed.
That failed.
So these variables
are probably okay.
What about this one?"
Okay, so you can get
a lot of information
from experiments that fail.
In fact, you absolutely to
be a successful scientist,
you need to learn how to work
with experiments that fail
because 90% of the
time, they fail.
Okay but you know, that's the
way life is so you learn as much
as you possibly can
and then you move on.
But to make strong conclusions
though, you need experiments
where both the positive control
and the negative control are
working as expected, okay.
Okay, good question,
B. Other questions?
All right, let me
show you an example.
Let's imagine that you wanted
to amplify some DNA sequence
using a technique called PCR.
Details not so important now.
Hopefully, you already
know what PCR is.
I understand it's taught
in high schools now.
If not, you can look
it up in the textbook.
If not, don't stress about it.
I'll talk about PCR later.
Later, you'll need to
know how this works.
For now, let's just use it as a
method for amplifying DNA, okay?
And furthermore, here's a method
for visualizing DNA
as bands on a gel.
And I know all of
you have done TLC.
This is kind of like TLC except
the bands are upside down, okay?
But it's more or less,
it's like upside down TLC.
It's more or less the
same technique that's used
to visualize compounds
except we're visualizing DNA
by running it through
an agarose gel.
Again, if that technique is not
familiar to you, don't panic.
We'll talk about that
later in this class.
For now, we have a
method for amplifying DNA.
We have a method for visualizing
the resultant DNA, okay?
Now, here's our positive
control.
It's the lane over here that's
labeled with a plus, okay?
So over here is a
set of conditions
that you know results in DNA.
And notice that there is a band
right, a big bright band, okay?
So that tells us that our
positive control works.
You have a sample of DNA
that you know should amplify
under that set of conditions
and lo and behold, it gives you
that nice bright band.
Next lane, the next lane are
the negative controls, okay.
So we don't see that same band.
Say that is missing
the DNA sample, okay?
We don't see that same
band so we don't have
to get worried about it.
Final lane, this is
our experimental lane.
Okay, you do these two
experiments, the positive
and the negative control just
to see whether your sample
over here is working, okay.
And here's the one that has the
actual test sample and notice
that it gives you
DNA and it turns
out the technique separates
on the basis of size.
It gives you DNA of a
different size, okay?
So we have both a positive
control that works as expected.
We have a negative control
that works as expected
and then we have our
experimental one.
In a typical experiment
in my lab, we'll have six
or seven negative controls and
maybe two positive controls just
so that we know what's going on.
We don't -- we cannot visualize
what's going on so we need all
of these controls to follow
what's actually happening
in the test tubes, okay?
Or sometimes even smaller
than test tubes, okay?
Sometimes, we're even down
on a single molecule level
so we really, really need
all these controls, okay?
I want you to be thinking
about these controls
when you design your proposals.
Okay, good proposals
will have both positive
and negative controls.
How you design your experiments
and how you discuss them
with me will in the end
determine how creative they are
and how robust they are
and how likely they are
to stand up to scrutiny.
Okay, if you want to propose
something that's totally wild
like I don't know, time
travel or something like that,
I will discourage you.
But let's say you want
to propose something that's
not quite so wild, okay,
but you come up with a
whole bunch of controls
that will really tell us
something about whether
or not your experiment is
working, I'll go with it, okay?
So be as creative as you
possibly can be, okay?
I'll look forward
to reading those.
All right, let's
talk about tools.
So the first tool that's
used quite extensively
in chemical biology
laboratory involves dyes
that are turned over.
These are these color-metric
indicators as they're termed
and have been used
for hundreds of years,
probably at least 120 years in
chemical biology experiments.
Okay, they're used for
all kinds of things.
They're used to stain cells.
They're used to follow
enzyme reactions.
And here is one example
of these dyes.
If you have some sort of
enzyme in your reaction
that you're trying to assay
and the enzyme somehow
cleaves this ether bond,
what will happen is this will
then release a nitrophenolate
molecule shown here.
This nitrophenolate is
a nice yellow color.
Okay, so you can
very clearly see.
This one is clear.
This one is yellow.
Okay, so everyone could
see that difference?
Okay, so if the enzyme
is present
and the enzyme is functional,
you get a nice yellow
color from this solution.
Okay. Now, this is
really powerful.
Okay, this gives you a way of
turning stuff that you can't see
into stuff that you
can then visualize.
Okay? And furthermore, this
is typically quantitative.
In other words, you can
pass light through here,
see how much light
gets absorbed --
say you pass visible
light through here --
see how much light gets
absorbed and use this
to quantify how much enzyme
is present in your solution.
Okay, doing this gives
you a really effective way
at addressing things
like enzyme kinetics, at,
you know, different properties.
You can look at say,
binding between receptors
and ligands using this
type of technique.
So, this is bread and butter
of chemical biology labs.
Okay, B, you have
another question?
>> [Inaudible] I know
that [inaudible] reaction,
so [inaudible] concentration
of enzymes [inaudible].
>> Okay, yeah.
So, B's question is how do
I know the concentration
of the enzyme in this reaction?
How do you make it quantitative?
Okay, so what you will do is
you'll have a series of controls
where you have a known amount
of enzyme that's turning
over this dye and then
you see how yellow it gets
after five minutes with that
known quantity of enzyme.
Okay? And then you can use that
to calibrate this experiment.
Okay. So -- yeah.
So there's subtleties to
everything I'm telling you,
but this isn't too hard.
Okay? Thanks for asking.
Other questions?
Okay, so in this example we're
looking at light that's absorbed
and then this absorbents results
in the molecule radiating
out the energy of the photons
that it's absorbing as heat.
Okay, in a different
experiment the light is absorbed
and instead of the energy of
the photons being radiated
out as heat, instead it's
blasted out by the molecule
as a photon with a lower energy.
Okay? So it has a
different wavelength
of light that's being given off.
Okay, so here's a series of
different molecules that have
that property in that
they absorb protons
and then radiate back out
photons of lower energy.
These are used in fluorescence
experiments extensively
in chemical biology.
These are used to visualize
molecules inside cells,
inside organisms, and in whole
hosts of different experiments.
Okay, so I already
told you this.
Flurorophores absorb photons
of light and emit a photon
at a lower wavelength.
Okay? You can select in your
microscope just those photons
at that lower wavelength
by setting up a filter.
Okay? So the way this works
is if your fluorophore --
let's say this fluorescein
over here.
So here's your fluorophore.
It's going to give you
this greenish colored light
and in your microscope
you will have a filter
that filters out
all other light.
Okay? So this prevents
back scatter --
except for light
of this wavelength
that is this nice green color.
That will give you exactly
where this fluorescein molecule
is binding inside the cell.
Okay? Furthermore,
this technique is
extraordinarily sensitive.
It's one of our most sensitive
techniques in chemical biology.
Supplanted only by the thing
that Miriam is working on.
Okay, so Miriam is doing
something that's going
to be even better.
But for now, up until say two
years ago, this was the champ
and you can get down
to single molecules
under the right conditions
using fluorescence.
You can actually see one
fluorophore fluttering away
as its releasing photons.
Okay? Pretty amazing.
Okay? I will tell you that
those right conditions,
completely non-trivial.
Okay? It takes a cooled
CCD camera that's very,
very large and very expensive.
This is not like your
cellphone that's hooked
up to the top of the microscope.
This is a really, kind of a
very special type of camera
to visualize this sort of thing
and pull up enough photons.
But in the end this is
really powerful stuff
because if you can visualize
just one molecule inside the
cell, then you can start
getting a processes
that really govern
how cells work,
where cells are oftentimes
responding to a lower number
of molecules inside them.
Okay? So this is a
really powerful technique.
It's used for all
kinds of things.
In this example I'm showing
you two cells that are dividing
and they're being pulled apart
by these spindles over here --
sorry, the DNA in blue
is being -- or in cyan --
is being pulled apart by
this spindle apparatus
into the two daughter
cells and the actin,
which is the protein scaffold of
the cell, kind of the skeleton
of the cell, is highlighted
in a red over here.
Okay? Absolutely spectacular,
stunning imagery really
that you can find examples of
where this technique is used.
This is completely ubiquitous.
This technique is used
for visualizing stuff
inside the cell.
It's used for visualizing
stuff outside the cell
and little tiny reaction flasks
for doing screens of drugs,
for doing phenotypic
assays of cells as well.
Okay, and question over here?
[ Inaudible Question ]
Yeah. So the single
molecule technique
that I described
would use a FRET.
So, thanks for asking.
Other questions?
Yes, over here?
>> So, basically --
>> What is your name?
>> I'm sorry, sir?
>> What is your name?
>> Chelsea.
>> Chelsea.
>> So, basically these
small molecules are made
so that it can bind to a
specific part of the cell?
>> Chelsea's question
is a really good one.
Okay, so Chelsea's asking, you
know, why should, you know,
this dye bind to
the DNA over here
and nowhere else
inside the cell?
Later we'll be talking about
the dyes that bind to DNA
and what makes them special,
but you're absolutely right.
They need some way of
getting guided into the cell.
So, for example, these
actin, the red color
of the actin I believe is an
antibody that binds to actin.
Okay? So that's a big
molecule that I showed earlier.
That antibody is then
attached to this rhodamine.
Okay, so rhodamine is
attached to the antibody.
The antibody that's being
used is specific for actin.
It binds to actin and it's a
professional binding protein
that was raised just to bind
to actin and now it's going
to highlight all of
the actin in the cell
in this rhodamine
red color over here.
Okay? Really cool stuff.
So, thanks for asking.
But you have to have
some other technique
that will target the fluorophore
specifically to what it is
that you're lighting
up inside the cell.
Okay? Great question, Chelsea.
Other questions?
Okay, so again, totally
ubiquitous technique,
used very extensively.
I imagine every single one of
you will have some experiment
in mind that will use
either fluorescence assays
or colormetric assays
of your molecules.
Okay, now here's the deal.
We can expand these up.
I've shown you two
different assays.
We can expand these up to
look at literally thousands
of molecules a day and thousands
of conditions a day using,
for example, micro titer plates.
Okay, so these are plates
that are about this big.
So, they're not that big,
and they're standardized,
and they have a standard
number of wells on them.
So the ones my lab uses are 96
or some sometimes
384 wells per plate.
That's this big.
But it's not unusual
to have 1536wells
in a little space
that's about this big.
Okay? Where each well is,
you know, say 10 microliters
or something like that.
Okay? But what that
means then is
on that plate you can
assay1536 different conditions.
Okay? So that's 1500
different conditions.
Okay, maybe 50 of those
are different controls --
negative controls,
positive controls.
But you're still
looking at a huge number
of different molecules,
of different --
other variables that
you're testing
in that one little, tiny area.
And it's not infrequent
for me to visit places
where they have a whole room
this size filled with robots
that are pipetting -- that's
this technique over here --
pipetting on an automated
fashion reagents
into these tiny little plates.
And then the robot has
like a little, you know,
arm that then brings
it into a reader
and absorbance is then
read out automatically
and all this data is
imported into your desk
and appears on your laptop.
Okay? Very cool isn't it?
Okay? So, yeah, it's a
great time to be alive.
Okay, so this absorbance we
talked earlier how it can be
used for quantitative analysis.
Oftentimes we rely on antibodies
to bind with specificity
to a particular molecule.
This is the question
that Chelsea was asking.
It's not unusual to us
to actually add an
antibody that's specific
for some target inside the cell.
Okay? And so we're going this
so that we can actually look
at just that individual protein.
And I showed you earlier
the structure of antibodies.
That structure allows them
to be very, very specific.
If an antibody is attached to
an enzyme then you can look
at turnover of a dye and that
can visualize the presence
of a molecule as
turnover of a dye.
Okay? Everyone still with me?
Make sense?
Okay, and the scope
of this is enormous.
Pharmaceutical companies
will screen
through half a million compounds
in two weeks using
techniques like this one.
Okay? And there might be
two humans that are involved
in those experiments, both
of whom are keeping the
reagents and the robot happy.
Okay? It turns out actually
programming the robot,
not as trivial.
So, you know, it's
very different
than telling the
undergraduates, "Okay,
I want you to pipette
all these things."
Okay, this is much
more industrial scale.
Okay, and it's used very
routinely in Chem-Bi labs.
Okay, sound good?
All right, let's move on.
Another very powerful technique
that's used quite routinely is
basically a Darwinian
evolution technique
where you can evolve organisms
that can accomplish
some chemical goal.
For example, over here
this is an experiment
to find mutant bacteria that
can take advantage of iron
and metabolize this iron.
So -- and this plate over here,
this left side is
the negative control.
These are bacteria
that you don't expect,
that were not mutated
and on the right side --
so you do not expect them to
be able to handle the iron --
and on the right side, these
little circles are examples
of the colonies of bacteria
that can take advantage of iron
and actually accomplish
their metabolism.
On the right side,
here's -- in B, panel B --
this is a different
experiment where you're looking
for bacteria colonies
that can produce lycopene.
Lycopene is the red dye
that's found in tomatoes.
It's the reason why
tomatoes are red.
And it also is thought to have
some anti-cancer properties,
although evidence for that
is not as well supported.
But in any case, you can
imagine evolving the bacteria,
putting in the genes that
encode lycopene production
and then evolving the bacteria
to produce this red-color dye.
And then at the end of
the experiment you'd go in
and simply pick out the reddest
of the colonies over here.
Now if you look closely at this
there's some really, really,
really interesting
stuff going on.
Okay? Do you notice how
some of these are kind
of mottled in appearance?
This one has some
little red dots
and then it looks mainly clear.
What's going on there?
That's absolutely fascinating.
Okay? I'd like to
know more about that.
So the essence of being good
scientists is not simply
running experiments.
The essence of being good
scientists is designing good
experiments and then observing
the results like a hawk.
Okay, you have to look at these
things intensely, intensely,
intensely and ask questions.
Why is there a white halo around
this one and then a red inside?
What is different
between the bacteria here
and the bacteria out here?
Maybe it's a trivial reason.
Maybe these guys have had more
time to produce their lycopene
and these guys are just,
you know, they haven't grown
as long on the outside.
But you still would
want to know that.
And so being a scientist is all
about designing good experiments
and then next observing,
observing, observing,
and making those observations.
That's where we make
progress in science
and where we make progress
in chemical biology.
Okay? Sound good?
All right.
Oh, I didn't tell you about
the Darwinian evolution.
You can imagine getting
a bunch of mutants,
picking out the winners over
here, mutating them again,
pick out the winners, mutate
again, pick out the winners.
That's the same process of
evolution that we talked
about on Tuesday where
you diversify the pool,
select for fitness, keep doing
the same thing again, and again,
and again, until eventually
you have some super growers.
Ones that can grow
really, really fast,
under those conditions.
Okay, and that would be really
interesting to understand
at a molecular level
what's going on there
and what's allowing
them to do that.
Okay, viruses are very powerful
tools for gene delivery.
They're very efficient
at infecting cells.
I'll be showing you
an example of viruses
in action in just a moment.
My laboratory grows large
quantities of viruses
as a tool for chemical biology.
Their major goal in life is
to make copies of themselves.
That's what they do.
Okay, they have a very
short lifetime and during
that time they are
totally fixated
on making new copies
of themselves.
Because they have such
short lifetimes and they're
so ruthless at amplifying
themselves this provides a very
powerful tool for selections.
Okay. Let me show you
an example of this.
The example is using a
technique called phage display,
which again is applied by my
laboratory and many others.
What we do is we start
with the filamentous virus.
Okay, so each one of these
little hairy things over here,
each one of these thread-like
things is a single virus
and the virus, this
particular virus infects e-coli.
So, like all viruses the inside
of the virus is an
encapsulation --
encapsulates genetic material.
In this case this
virus encapsulates DNA.
There's other viruses
that are RNA-based.
This one happens
to be DNA-based.
Okay, now here's the great part.
As a chemical biologist,
we can go in
and manipulate the DNA that's
found inside the virus.
When we do this, we
can coax the viruses
into producing large
numbers of different viruses,
each one with a different
protein displayed
on its outer surface.
Okay? Each one with a different
protein outside, on its outside.
Okay, that's called displayed.
Okay, and then you
can do selections.
So for example, you have, say,
a billion different viruses,
each one with a different
protein displayed out here.
You can then throw these viruses
at a chemically modified surface
down here, and then simply
take out the winners,
the ones that can grab
on to this chemical found
on the outer surface over here.
Everything else that can't
grab on is washed away.
You wash this away using
some sort of buffer.
Okay, so you just flow water
over this for five minutes.
I guarantee you, everything
that's a weak binder,
everything that can't
really get a good grip
on the chemically modified
surface gets thrown
in the trash.
Okay? And then you start
amplifying up those winners,
and then you do the
process again,
and then you do the
process again, and again,
like four or five times.
By doing that you start
to get very tight binders
to this chemical found on the
surface that you're targeting.
Okay? So, this is
a way of starting
with literally 10 billion
different molecules and coming
down and identifying just the
few that do something special,
such as bind to this
chemical over here.
Okay? Question over here?
>> Seeing the virus so
small, how can you pick
out every single virus?
>> Yeah, yeah.
Okay, that's a great question.
So how do you even
manipulate these viruses?
So what we do is we infect
back their e-coli hosts
and then we can make colonies of
those e-coli that are infected
where each colony has
one and only one type
of virus inside of it.
Okay? And then you can
actually see the virus there.
Okay.
>> [Inaudible] virus that can
attach to the [inaudible].
>> Yeah. Yeah.
>> [Inaudible] virus
to the e-coli.
>> Yeah. Let me show
you on the next slide.
Okay? Great question.
Okay, so the question
is about the particulars
of how this technique works.
Again, here's the
viruses over here.
Here's the size of our library
that's around 100 billion or so.
That's the maximum
size that we can make.
Notice that in this
electron micrograph
over here there is a
little cluster of grapes
at one end of the virus.
That's its head.
That's what it uses to
grab onto the e-coli
that it's going to infect.
Okay? So, that's
this part up here.
Okay? That's the head of the
virus, that cluster of grapes.
And again, the DNA is stuffed
into a long pipe of virus
over here, and the
virus is very flexible.
Okay, so this virus
is like a hose
in terms of its flexibility.
Okay? Now, here's the experiment
that I was getting
asked about earlier.
So what you do is you make your
library of different viruses,
each one with a different
protein displayed out here
and then you throw those
viruses at some target.
Pac-Man. Okay, this Pac-Man
shaped target that happens
to be stuck on the surface
-- on some sort of surface.
Okay? You then select all of
the things that bind to Pac-Man
and wash away everything
that doesn't bind.
Okay? So in this step
you go from 100 billion
down to just say --
let's say a couple 100.
Okay, and then you pick out
these viruses, you amplify them
up in their host, e-coli,
and then you do this again.
Okay, so again, we
target Pac-Man,
wash away the non-binders,
amplify up the binders,
wash away the non-binders,
amplify the binders,
and you just keep doing this
a bunch, a bunch of times.
Okay? At the end of it
you'll end up with say --
let's say 50 to 100
that bind really well
to the targeted PacMan
shaped molecule.
Okay, so now you want to
go in and you want to look
at those individuals and see
which one binds the best.
I think that's your
question, right?
Okay, so what you do is
you infect the winners
into E. Coli -- this
is a bacteria --
and then you can
plate out bacteria
such that you end
up with colonies.
Okay? That was shown over here.
Each one of these dots
is called a colony.
These are genetically
identical bacteria.
In the case of virus
infected bacteria,
each one of these colonies will
have a different virus in it --
a different bacteria
phage in it.
Okay? And then you can assay
each one of those individually.
Okay, it turns out that this
principle of vast library
of proteins that are displayed
on phage is also
applicable to DNA and RNA.
And this is another tool
that's used routinely
in chemical biology
laboratories.
So my colleague, Professor
Andrej Luptak, for example,
routinely makes huge libraries
of RNA and then selects
for binders from
this big library.
So here, for example, is
a derivative of rhodamine,
a molecule that I
showed you earlier,
and here's an RNA
sequence that likes to bind
to this rhodamine-like
molecule that I showed earlier.
So you can select for binders
to all kinds of different things
from these vast pools
of both DNA and RNA.
Okay, using exactly the same
principle that I showed earlier,
you attach this molecule
to some surface,
you throw at that surface
the big pool of say, RNA,
wash away all the non-binders,
grab onto the binders,
amplify them up,
repeat the process.
Okay, so it's simple,
molecular evolution.
Okay? Exactly like the evolution
that we talked about on Tuesday.
Now the reason why
this is important --
it's important to apply this
evolution is you cannot know
in advance exactly what
sequence is best going to bind
to some complicated
molecule like this.
Okay? I know it would be
really cool if I could sit
down with laptop and, you
know, crunch some numbers
and at the end of that get
the perfect RNA sequence.
But we chemical biologists
can't do that.
Okay? We just don't know
what are the design rules
for designing something that
has a pocket shape like this.
And furthermore, what are the
functionalities that we're going
to need that'll be complimentary
to the partial positive charge
over here, on the
lone pairs on oxygen,
the [inaudible] over
here, et cetera.
It's better just to go
out and do the experiment
and just see what you get,
and then analyze what
you get at the end of it.
Okay, make sense?
Okay. So that was an
example in your tool kit
of using libraries
both on phage,
libraries that are DNA or RNA.
The next thing in your tool
kit are small molecules.
So small molecules are used
extensively in chemical biology.
So some of these
molecules are antibiotics.
Some of them are natural
products that are found in --
that are being produced
by microorganisms
as they fight off
their invaders.
But others are discovered in
chemical biology laboratories
with a particular function.
Okay? And so these molecules
are used quite extensively both
in chemical biology laboratories
but also in Cell Biology
and in biochemistry labs.
So, for example, yesterday
I showed you the pathway
of the central dogma, which
is the information pathway
for biosynthetic
information inside the cell.
Small molecules, such as
the one shown over here,
are known to inhibit pretty
much every step of this pathway.
And so, on the shelf you can
have molecules that would say,
disrupt the process
of translation,
like cyclohexamid, shown here.
Or other molecules that
disrupt transcription,
such as alpha-Amanitin,
shown here.
And these are molecules
that you can buy
from your chemical supplier.
Okay? So these small molecules
give you tools to shut
down specific events
inside the cell.
Okay, now what's so powerful
about this is you can control
the dose, the location,
the time of delivery, et
cetera, with perfect control
over those type of things.
Okay, the dose is simple.
Right? You add the
exact concentration
of the small molecule you want.
And, where this is important is
that also controls the percent
of inhibition that you're doing.
Okay, so let's say you want
to shut down a little bit
of protein translation but
not all protein translation.
Maybe you don't use
a huge quantity
of cyclohexamid over here.
Maybe, more likely though,
you just want to shut
down all protein translation,
so you add a large
concentration of cyclohexamid.
In addition, you can
control the location.
So you can deliver the
molecule to some space.
Let's say you're looking at
an organ under the microscope
and you want to know, you
know, what happens if I shut
down protein synthesis on
this part of the stomach,
but not this other
part over here?
You can dose that
part of the stomach
and leave the other
part undosed.
In addition, you can
control the time of delivery.
Right? You can say, look at --
if you're looking at
circadian rhythms inside --
I don't know, inside
your neuro cells.
Right? Circadian rhythms are the
timing of clocks that is used
by organisms to coordinate
their day.
You might be really interested
in knowing what happens
if I shut down transcription
at --
right before the
organism goes to sleep?
So being able to add the small
molecule at a precise time,
in a precise location,
with a precise concentration is
really powerful, and it's one
of the reasons why
small molecules are
so important inside cells
-- inside chemical biology
and cell biology labs.
Okay, any questions about
what we've seen so far?
Okay, I've shown you a whole
series of different experiments
that you can do and
you can plan to do.
I want to show you next the
players that you're going
to be using for designing
your proposal ideas.
Okay, you're going to
be using model organisms
because as I told you
earlier I don't want you
to plan experiments on humans.
Okay, that would not be
the point of this course.
Okay? Instead, what I'd like
you to use is model organisms
or samples that are obtained
from consenting human adults.
Okay [laughter]?
Okay, so in general though,
when you're choosing a a model
organism you want to choose one
that grows easily, that's easy
to study, that grown quickly,
and has some relevance
to human biology.
Okay, not every model organism
is going to be so great.
If you want to study,
say, you know,
the hearts of Burmese pythons,
and Burmese pythons take years
to grow or something like that,
it light be a very long PhD
for you or your students,
and no one likes that.
Okay, so you want to choose
organisms that grow quickly,
that are inexpensive to grow,
that don't require really
exotic conditions to grow.
You know if you have to feed
your Burmese python rabbits
every two weeks or
something like that it's going
to be expensive and it's also
going to be a lot of hassle.
And so you need to have
some really good reason
to have chosen Burmese
pythons as the model system.
In general, these are the
model systems that we use
in chemical biology
laboratories,
with the exception
of humans down here.
I'm just listing this for
a point of comparison.
Okay, so I will step through
each of these and tell you
about their properties.
Okay? So for example,
I've shown you earlier use
of this bacteriophage.
This is a virus that only
effects E. Coli bacteria,
hence the name bacteriophage.
So it's a virus that eats --
phage means to eat -- bacteria.
And this only affects E. Coli.
This makes it very convenient
for us to use in the laboratory
because we don't have to
worry about if it "escapes."
We don't have to worry about
it infecting my co-workers,
the graduate students,
the post-docs in the lab.
Furthermore, it has
a very simple genome.
It just has 11 genes
in its genome.
That makes it easy
to manipulate.
Okay, this reference here is to
the picture that I'm showing you
and I showed earlier
in the class.
Okay, it's the lecture
on [inaudible].
In addition it grows in E. Coli.
Let me show you what
E. Coli look like.
So here are E. Coli next
to a red blood cell.
Let's see, is this right?
No, sorry, this is
next to a macrophage.
So these are the cells in your
immune system that are charged
with eating E. Coli, okay,
or other foreign invaders.
Okay? So each E. Coli is on the
order of about one micrometer
in scale and each human
cell is on the order of 20
to 30 microns in scale.
Okay, so that gives
you kind of an idea
and I think this picture
dramatically illustrates the
relative scale.
This makes sense, right?
E. Coli are prokaryates.
I showed you structures
of prokaryates last time.
Human cells, of course,
are eukaryotic cells.
They're a lot more complicated,
they have a lot more organelles
inside them, et cetera.
Okay, so classic experiment
in biological history.
This was -- this is
Griffith at the top --
that's Fred Griffith at
the top with his dog Bobby.
I always like to know the
names of scientists' dogs.
Fred Griffith learned to
recognize R pneumococci
and differentiate them
from S pneumococci.
So R equals rough,
S equals smooth.
And he found that dead S
pneumococci could transform live
R. And Avery, this guy down
here, working at Rockefeller,
showed that if you
isolate the DNA
from the dead S bacteria it
could transform the R bacteria
into S. Okay?
So, the important idea
there is that it showed us
that DNA was the
hereditary unit of the cell.
That DNA was encoding the
machines inside the cell
that were making the outer
surface either smooth or rough.
Okay? Sad history here,
Fred Griffith died
when the Germans
were bombing London.
He died in the London Blitz.
Okay, so E. Coli
extensively, extensively used.
I showed you a couple
of examples,
including phage display today.
Yeast are used as a model system
for a very simple eukaryote --
as a -- you know, equivalent
to the prokaryotic E. Coli,
but very simple to grow,
very easy to genetically
manipulate, et cetera.
As things get more complex
we get towards organisms
like fruit flies over here.
Fruit flies are used
extensively in laboratories
because they grow quickly and
you can do selections for things
like morphology, shapes of
wings and things like that.
But then even more complex
traits such as behavior.
And I will show you
one example of this.
This is one of my all-
time favorite examples.
This is the great Ulrike
Heberlein, a professor at UCSF,
and in this experiment the
Heberlein lab has built an
apparatus that they
call an inebriometer.
Okay, so this looks
at drunk fruit flies.
Okay, so here's the
way this works.
This bottle over
here contains ethanol
and then she pulls a little
bit of a vacuum on this
so that the vapors -- or she
blows the air over the top
of this so that vapors of
ethanol come off over here.
And then she applies a bunch
of different fruit fly mutants
to the very top of the column.
Now when fruit flies land
on these cones over here
and the cones are made
out of like a little wire,
the fruit flies grab
onto these things.
Okay? That's what
fruit flies like to do,
they like to perch on things.
But now they're being washed
over with this ethanol vapor.
Okay? So the alcohol
is coming over them
and they're inhaling it.
They can't get away.
And so as they start to wobble
back and forth they fall
down to the next cone, and
then they grab on again.
But then they start wobbling
around as they get drunk
from the ethanol and they
drop down to the next one.
Until eventually down here
they totally pass out.
Now, the wild type fruit fly
over here takes 20 minutes
to come through this column,
whereas there are mutants
that the Heberlein laboratory
found that only took 10
to 15 minutes to get
through the column.
In other words, those were fruit
flies that were getting drunk
and passing out faster
than the other fruit flies.
So the chemical biology part
of this experiment would be
to understand what genes are
involved and then at level
of [inaudible] bonds why those
genes are making the fruit flies
drunk faster.
Okay, now I do have one request.
Please do not plan your
chemical biology proposal using
an inebriometer.
I have seen every
variance of this.
With marijuana smoke, with
all kinds of, you know,
things that cause all kinds
of interesting effects.
So use any other experiment.
But what I like about this is I
loved the experimental design.
It's very straightforward.
Any one of you in this
classroom could've invented that
and that's what I'm going to
be looking for when I look
at your proposals
later in the quarter.
Okay, I'll see you
a week from today,
back in this lecture hall.
We'll be talking about
more model systems
and then we'll be
talking about [inaudible].
[ Inaudible Conversations ]
------------------------------8a15c2c9f526--
