So, let me now go over some of the
factors that affect the detectability of
GFP, and this is one of the problems that
people working in GFP used to have a lot
of trouble with. And even now if you were
a student trying it for the first time
on a protein that hasn't already been
worked out, you may find difficulty in
getting your signal. And people throw up
their hands. "What's wrong with my
experiment?" Well if you analyze it,
there's a whole bunch of factors. And
here the four major categories, which
we're then going to break down. The first
is you just got to make enough GFP.
GFP is not infinitely bright. The next is
that having made the protein by protein
synthesis off the ribosome - presumably
that's how it's genetically encoded - you
then have to make sure that it does a
good job of post-translationally making
that fluorophore. The third category is the
molecular properties of the mature GFP.
And this is the most intrinsic factor
that takes a protein engineer to fix and
the one that say things like S65T-
improved. And nowadays this is less of a
problem because people typically know
how they can acquire either from their
friends or go out and buy a pretty
up-to-date GFP that's already been
optimized in this. And finally, you have
to remember that you have to detect
these signals in a competition with
noise and background and there is noise
and background and fluorescence. A lot of
the rest of this microscopy course will
be teaching you about how to optimize
that balance. But a few things can be
said even now. So, let's go back to total
amount of GFP. Obviously, you've got to
have enough copies of the gene and
duration of expression, and this is back
to your traditional molecular biology.
You have you also have to have a strong
enough and promoter and enhancer. The
translational efficiency has to be
correct and that includes the so-called
Kozak sequence, which is the sequence
right up at the front of the coding
amino acids, which has certain optima to
help the ribosome get started. It's
helpful if you have the correct codon
usage. If you, the original GFP, of course,
had jellyfish codon usage.
Now you can get genes which
have been optimized for mammalian
or bacterial and so on. And there was a
time in the old days when people had
trouble with it because they were using
the wrong codons in their nucleic acid.
And you also want to avoid mRNA splicing.
It turned out that plants could find a
splice site within the jellyfish gene
and splice it out and of course then you
didn't make a functional protein. Most
these problems now are less important
because so much engineering and
experience has been done. How about the
efficiency of post-translational fluorophore
for formation. Originally, if you tried to
express this heavily in bacteria,
especially if you did it warm, the
protein would just crash out and
denature because it didn't like being
made in such quantities. And if you have
chaperones available to help fold
proteins better that helps. In fact, this
has even been used as a bioassay
internally for the presence of
chaperones. Pick up a crummy GFP, it has
trouble folding and see whether you can
make it fold. And that may be dependent
on the chaperone that you want to assay.
A lot of the times when you fuse this to
a host protein, that fusion has some
unfortunate properties very hard to
predict and the protein just doesn't
like to express. And sometimes people
already know. My favorite host protein
just won't tolerate you putting
anything on the N-terminus. Well then
don't be surprised if you try to put GFP
there, it won't like it. Sometimes we
don't know that in advance. And commonly
if the N-terminus doesn't work try the
C-terminus. GFP can work at either one in
general. But for any given protein you
don't know until you try it, and even if
they've poop out and don't work for you
you can even try to put GFP, insert it
within a loop of, or a comfortable spot
in the host protein. Sometimes that
solves the problem. Of course, at this
stage you're doing a lot of work of
trial and error but that's what it may
take. And of course, you do have to give
it time. Those post-translational
reactions in those complicated sequences
that I showed you in the ---
with asparagine glycine, all that takes
time. We now have some that can go pretty
fast and do it in only a few minutes, but
originally it took four hours from the
original jellyfish. So that's been
improved a lot by further
engineering. And as I said, we now have
fairly warm temperature tolerant
versions. As I said, you must have oxygen
present at least during the maturation.
Once the protein is mature you don't
have to have oxygen still there. The GFP
works just fine but it did need it just
that once, once per each molecule. So, then
the molecular properties. And we want to
improve the wavelengths of excitation
and emission, make them ones which are
easily detectable. And there'll be a lot
more on that in this lecture. We want to
improve the extinction coefficient, which
is the ability to absorb light, and
fluorescence quantum yield, which is once
you've absorbed a light how probable are
you to reemit it. GFP now is actually
pretty good. The good GFP, the best ones
now, have quantum yields of seventy
to eighty percent which is, and
theoretically you can never go up above
a hundred percent. So, there's not much
room left to improve that one. But we are
all still very interested usually in
preventing photoisomerization or
bleaching unless we specifically want a
trick that it requires bleaching GFP
sometimes, and I'll briefly mention some
of those. But most people, for most
applications, just want thing to shine as
brightly as long as possible. And this is
still an area of active investigation,
how to improve that. And I mentioned
importance of knowing whether it's
dimerizing, and in some cases getting rid of
it. Particularly when you are fusing it to a
host protein. Finally, competition with
noise and background signals. You must
remember that a lot of the culture media
are also fluorescent at similar
wavelengths. Particularly if they have
riboflavin, if they have a lot of serum
proteins and so on. And that will
generate a haze of fluorescence that has
nothing to do with GFP but will obscure
small amounts of GFP. So, you want to get
rid of those if you're ever having a
detection problem. The location of GFP
actually matters somewhat, particularly
if you wanted
the fewest copies of GFP you want to
localize it into a tight little spot,
ideally a micron or so in size, preferably
where you know an advanced way to look.
And then you only have to ask do I see
it, a dot there, or do I not? And at that
level people can often see just a few
copies. And in the very best cases it has
been proven you, in not very general
cases, but you can occasionally see just
one copy of GFP. Though typically, you're
using so much laser light that you
bleach it very very quickly. So, compared
to that if it's just diffuse, it's much
harder to see the difference between
diffuse GFP and the diffuse background
that cells already have. They already
have autofluorescence even when the
culture media is very clean. You need to
have very good filters, which don't
overlap. And a lot of people who are
first starting fluorescence microscopy
just pick any filters they got around
them, that happened be rattling
around in the drawer underneath the
microscope. And those are often not
optimized and we need ones which
accurately excite the GFP but do not
enable crosstalk. And crosstalk is when, say,
a little bit of the excitation light can
leak through the emission filter, so
scattered light then instead of
fluorescence can contribute. And that's
something you just got to buy better
filters and dichroic mirrors. Finally, of
course, we need good detectors. Your eye
is actually quite an inefficient
detector and nowadays it's just a matter
of spending money on a sufficiently good
camera. And I'm sure other people in the
course will tell you a lot more about.