I think it's a truism in science that if you can see something,
you can study it.
What's really exciting about GFP is it has allowed us to see so
much within the cells that we were not aware of before.
When I heard about GFP in that seminar in 1989,
I was very excited about it.
I think I was the only person in the audience that was working on a
transparent animal,
the nematode Caenorhabditis elegans,
and at the same time interested in knowing where genes were expressed.
And I realized that if we could get the DNA for GFP,
we could insert that into the genes we were interested in.
And now those genes would cause the production of GFP and
we could see that in the living transparent animal just by shining blue
light onto the animal and seeing which cells turned green.
So the first experiment was to actually try to see,
could GFP be made in something other than the jellyfish?
At the time,
most of the people working on GFP,
they all thought this was going to be impossible.
But the person who actually did the first experiment in the lab was Ghia.
My name is Ghia Euskirchen.
I was a rotation student in Marty Chalfie's lab. When Marty called me up and
presented this fluorescence project,
I was actually really excited because for my master's thesis worked on fluorescence and he
thought that the project with GFP might be a good match and
in general,
who doesn't like fluorescence?
It's really beautiful and it's just fun to work with.
So from the very start,
what I wanted and what Douglas wanted was to be able to
show that GFP could actually form a fluorescent
protein,
one that we could shine blue light on and see green
in other organisms.
Mostly,
I wasn't really expecting the experiment to work,
one of the big reasons being because none of the other graduate students in the lab really wanted to
touch it.
They kind of had looks on their faces like,
yeah,
good luck with that.
The problem came in, in that all the people in the field knew that
the final protein had this arrangement,
this change of the cyclization of the backbone, that
is the production of that five atom ring.
And they were perplexed about this because to see a
chemical change in a molecule, to them, implied
that there had to be a converting enzyme.
And so they had convinced themselves that that was the case.
And so this was really a fool's errand to just put the
DNA encoding the GFP into an organism;
it would not work.
We decided to just try this anyway.
I think sometimes it's probably a very good idea to be just willing to try the
experiments and not be convinced by everyone's reasons of why those
are sort of silly to do. Being new to molecular biology,
I thought this will give me a chance to learn molecular biology so I was willing to try it.
And plus,
I could bring some fluorescence experience to the problem and hopefully
have something to contribute that way.
So the first experiment was to actually try to see,
could GFP be made in something other than the
jellyfish?
And by far the easiest way of looking at this would be to put it into bacteria.
And so that was the first experiment. When Ghia actually
started to do the experiment,
there were obviously several steps that needed to be done in order for this to work:
isolate that the original DNA;
make many copies of it by polymerase chain reaction;
take that DNA and transform or add that
DNA to bacteria so that each bacterium got one copy of
the DNA;
grow up those bacteria and look at it under a microscope that would
allow you to see fluorescence.
The first was to take the DNA that Douglas Prasher had
provided us and to make lots of copies of it.
And I wanted her only to use the coding sequence,
but the original DNA that Douglas had given us,
had not only the coding sequence but some extra
jellyfish DNA on either side.
I just didn't want to have anything extra,
I just wanted to have what we needed.
So we used the method of polymerase chain reaction, PCR,
which starts by having pieces of DNA, primers, that will
bind to specific parts of the DNA and allow that
part of the DNA to be copied and amplified so that you get many
copies of it.
So we were careful in the sense that we just wanted to use the coding
sequence. In 1992 when Ghia did these experiments,
this copying of the DNA using PCR
was prone to errors.
There were lots of ways that this could go wrong.
And so not all the DNA was a perfect copy of something.
And once it wasn't a perfect copy, then when it was copied,
maybe that same mistake would then be propagated.
This didn't bother us at all,
and the reason it didn't bother us is we were going to put all the individual copies
into different bacteria.
So if a bad copy got in,
maybe the protein wouldn't be made,
but enough of the bacteria would have good copies,
and if the product was going to be fluorescent,
those bacteria should be fluorescent.
The next part was inducing the E. coli to express the protein
and had two samples.
One were E. coli with the plasmid,
also known as a vector,
which had the GFP insert, and then another tube of E. coli that only
had plasmid,
no GFP insert.
But it was a little fuzzy because no one had really done this, had really looked at GFP outside
its native jellyfish.
It was important to have that control of E. coli that weren't expressing
GFP and so you could see that there was a difference in fluorescence between the
two. I was fairly stunned at the time because the whole experiment wasn't supposed to be that easy.
So it was really surprising it actually worked. And so
that first day in October,
about a month after she started graduate school,
Ghia was able to look down the microscope and actually see bacteria that were
glowing green when they were stimulated by blue light.
I found out several years later that at least three other groups
tried to do the same experiment.
This shows you that no one has a lock on any one idea.
Lots of people come up with a lot of interesting ideas.
But all these three other groups did something different from what we
did.
What they found is they needed to cut that DNA
to be able to add it.
And so they used enzymes, called restriction enzymes, to cut at very
specific places.
Now,
the enzyme that they used
did include the coding sequence,
but it also included extra DNA,
and no one actually knows why,
but those extra pieces are a disaster.
Having those extra bits of DNA prevent the production of the
fluorescent product.
So we were very fortunate because we didn't have the extra bits.
Our experiment worked the first time, we had green fluorescent
bacteria.
They put their carefully produced
DNA into organisms,
it didn't work.
And you'll remember that if it doesn't work,
maybe that's not because they've made a mistake,
but maybe because there's that elusive converting
enzyme that's needed to make the final fluorescent product.
Their conclusion might have been, probably was, oh,
GFP would have been a great thing,
but it needs something else for it to work.
Our conclusion was,
it doesn't need anything else,
it works completely on its own,
it's going to be a very useful way of marking cells and proteins.
What Ghia's experiment told us was that the idea
of having GFP as a marker worked and that gave us hope that we
could now put it into other organisms,
particularly into the nematodes Caenorhabditis elegans,
the animal that I was working with, so we could start to look at cells.
One of the most interesting aspects, more or less sociologically than anything else,
for me has been to see how this rather simple
experiment of putting GFP into bacteria
and worms has expanded as everyone else has added
their thoughts and ideas to using this protein.
It was estimated in 2014, by the people at Chemistry
Abstracts, that in the twenty years since our paper was published, that there
have been on the order of 160,000 scientific papers using
fluorescent proteins.
And the sheer range of the things that people have
tried, thought of, has been really remarkable.
That first day when I was listening to the seminar
where I first learned about GFP, I thought about some experiments that I would like to do,
but my ideas have been dwarfed by all the really incredible ideas that people
have had coming out of this.
Sometimes people ask me,
did you think this was the type of experiment that would win a Nobel Prize?
And I can honestly say that I wasn't thinking about prizes or anything,
I was more excited about the experiment.
One might ask the question,
why would someone interested in developmental biology, in
genetics,
how did I wind up being the person that introduced this
very useful tool into biology?
And I don't have a very good answer to that, other than the fact I was just excited when
I heard that first seminar about GFP and realized it would be a very,
very useful tool.
And I thought it would be a lot of fun if we did those experiments.
I think that's actually a very important part of science, is that we're always
working on the edge of what we know.
We're not trying to duplicate things that we already know,
that doesn't give us more information.
We're trying to understand something new.
We're not all that great at understanding things that are new.
So it's not maybe a surprise that experiments don't work,
but we keep trying to go after it,
and once we do have those discoveries, or those breakthroughs,
it's exceptionally rewarding.