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A TD-700 Laboratory Fluorometer
Method for
The Assaying of Green-Fluorescent Protein
in Whole Bacteria
Introduction
The green-fluorescent protein (GFP) has
become an extremely exciting and useful marker for gene expression. This
note describes a method to assay whole living E. coli cells for the presence
of GFP using the Turner BioSystems TD-700 Laboratory Fluorometer. It will
arrive at an estimate of mature GFP in viable cells collected from liquid
broth.
This is different from quantitating GFP
in cell extracts and at various stages of purification. One of the problems
encountered when measuring whole cell suspensions is that such solutions
are extremely turbid. This has the effect of scattering the excitation
light in the solution thereby reducing the amount of light reaching the
GFP chromophore. In addition, light scattering may contribute to false
emission readings. To overcome these problems, we calibrated the TD-700
against a GFP concentration curve in cell solutions that do not produce
GFP, and in solutions prepared and read in a frosted 13mm x 100mm borosilicate
cuvette. One of the drawbacks with measuring GFP in whole cells as it
is being produced is that its chromophore forms slowly in the presence
of molecular oxygen (O2). As a consequence, any direct measurement is
probably an estimate of the total GFP present since a portion of the GFP
in whole cells has yet to become fluorescent.
2. Materials Required
- TD-700 Laboratory Fluorometer with standard
PMT (P/N 7000-009)
- Near UV Mercury vapor lamp (P/N 10-049)
- 13 x 100 mm round test tube holder (P/N
7000-981)
- Optical filters providing excitation
at 390 nm (P/N 034-0390) and emission at 510-700 nm (P/N 10-109R-C)
- 13 x 100 mm borosilicate glass test
tubes (P/N 10-031)
- PBS (Phosphate Buffered Saline) 20 mM
phosphate (PO3)4 with 150 mM sodium chloride (NaCl), pH 7.4
- 20 µg solution of recombinant Aequorea
Green-Fluorescent Protein from ClonTech Laboratories, Inc. [catalogue
# 8360-1] Telephone (650) 424-8222
- Frosted Cuvette: The lower 3/4 of a
13mm x 100mm borosilicate test tube is sanded thoroughly on the outside
with #60 Aluminum oxide sand paper (dry), followed by a thorough sanding
with #320 silicone dioxide sand paper (wet), to achieve a frosted effect.
3. Fluorometer Calibration
3.1 Set-up
Upon receiving and unpacking your TD-700,
prepare it for GFP quantitation.
Follow the procedure outlined in your
operation manual to install the near UV mercury vapor lamp.
Install your GFP filters into the filter
cylinder and place it into the fluorometer (remember to wipe any fingerprints
off of any filter or cuvette after you handle them). The ports for each
set of filters are labeled EX for excitation and EM for emission and
each pair of ports is labeled A through D. Choosing one pair of ports,
carefully insert your filters. The 390 nm filter has a reflective face
which should be installed so that it will face out towards the lamp.
Each filter is held in place by a circular rubber grommet, or o-ring.
At the ends of the filter cylinder are
labeled marks corresponding to the pair of filter ports you have chosen.
Insert the filter cylinder into the fluorometer while aligning this
mark with the silver alignment mark found on the inside rim of the fluorometer's
sample chamber.
3.2 Calibration
Close the TD-700 lid and turn the unit
on. It will count down 600 seconds to warm up.
After the instrument warms up, insert
the cuvette holder into the sample chamber. Note that the top of the
cuvette holder has an arrow shape molded to the top part of the holder.
Orient this arrow pointing toward the silver alignment mark on the inside
rim of the sample chamber.
You will be performing a multi-optional
mode calibration (refer to page 21 in your manual if needed). Press
[enter] on the keypad. Enter [1] on the keypad to enter SETUP, then
press [1] again to enter MODE. Using the ARROW key to choose the mode,
select MULTI-OPTIONAL then press [ESC] to return to the setup menu.
Enter [3] to enter the units menu, then
use the ARROW key to select µg/mL. Press [enter] to return to the setup
menu.
Enter [2] to enter the calibration procedure
menu, and select DIRECT CONCENTRATION using the ARROW key. Press [enter],
then [ESC].
Enter [2]. Enter the Max Range you want
to use (the default range, 999.9, is fine). Enter [1] to select the
range given, or [9] to change it. When you select [1], you will be queried
for the number of standards you will be using (between 1 and 5). The
suggested dilution range for this method is 5 µg/ml to 1 µg/ml (i.e.
five standards) in buffer using a frosted cuvette or in a non-GFP expressing
cell suspension, in PBS, in an unfrosted cuvette. Enter the number of
standards you will be using, then press [enter] and insert the first
sample to be read. Make sure the volume in the tube is over 3 ml, so
it is above the window of the cuvette holder.
The fluorometer will ask you if the
concentration value is correct. If it is, enter [1], if it is not, enter
[9] and input the correct value. When you select [1], the machine will
prompt you to select the [*] button to store that sample into memory.
The cycle will repeat as it asks you for the next highest standard.
When you finish calibrating the lowest
standard in your series, you will be prompted for your blank, which
will be your cell suspension or buffer solution without any GFP. Place
that in the TD-700, and press [enter]. The TD-700 will zero itself.
When the value at the right of the display becomes stable, enter [0]
as requested. It will record your blank and indicate that the calibration
is complete.
The TD-700 will give you a direct concentration
value when you insert your samples and close the chamber door.
4. Quantitating GFP In Intact Bacteria
4.1 Place the sample of E. coli
suspension to be assayed for GFP into a 13 mm x 100 mm borosilicate test
tube (frosted or unfrosted). If your cells were cultured in media possessing
residual autofluorescence, such as LB broth, you can remove it by pelleting
the cells using centrifugation and resuspending them in PBS. Always make
sure the final volume in the tube is over 3 ml (as the meniscus of the
sample will affect your readings).
4.2 Insert into TD-700 chamber and close
the chamber door.
4.3 Press [*] to begin reading. Record
reading when display reads END.
4.4. Example Quantitation
You have a strain of E. coli
expressing GFP under the control of the lac promoter which was grown
in a 55 ml liquid culture under induction overnight. Simultaneously
prepare your standard solutions by growing and preparing untransformed
E. coli under the same conditions.
Pellet 50 ml of the cell suspension
and wash several times with 50 ml PBS. Resuspend the cells in 5 ml PBS
(which will increase our readings 10-fold).
Perform the above mentioned calibration
and quantitation procedure (sections 3 and 4) to estimate the concentration
of GFP in your transformed cells.
Comparison of typical GFP concentration
estimates in this example for frosted and unfrosted cuvettes, after
considering your 10-fold increase in readings:
Tube Type = unfrosted, Estimated Concentration
= 0.89 µg/ml
Tube Type = frosted, Estimated Concentration = 0.84 µg/ml
These numbers are very similar and
reflect an estimation of the concentration of mature GFP present in
these cells. They also indicate that using a frosted cuvette instead
of suspended cells to calibrate the fluorometer might be a workable
alternative when you need to quickly estimate the amount of GFP that
is present in your cells.
5. References
A suggested method for the quantitation
of Green-Fluorescent Protein. Turner BioSystems Application note. 1996.
Davis, D. F., W. W. Ward and M. W. Cutler.
1994. Post-translational chromophore formation in recombinant GFP from
E. coli requires oxygen. In: Bioluminescence and Chemiluminescence,
Fundamentals and Applied Aspects. A. K.Campbell, L. J. Kricka, and P.
E. Stanley (eds.) John Wiley & Sons, pp. 596-599.
6. About the Authors
This GFP application note was written by
Daniel G. Gonzalez M.S., who is currently a Biochemistry Ph.D candidate
working in the laboratory of William W. Ward at Rutgers University (New
Brunswick, N.J.). His interests include the physical characterization
of chromophore formation in a variety of Green-Fluorescent proteins from
various organisms. He was assisted by John Covalesky, who has recently
graduated from Rutgers University with a B.S. in Biochemistry. William
W. Ward is one of the pioneers in GFP research and is still very active
in the field. He and Daniel currently use a wide assortment of techniques
in fluorescence analysis, protein purification and molecular biology to
study and characterize Green-Fluorescent Proteins. The Ward lab prepares
and coordinates a series of short courses in biotechnology that features
Aequorea GFP as a model protein for purification and molecular manipulation.
Information on these courses can be obtained by contacting Daniel:
Phone: (908) 932-9071, ext. 219
E-mail: meton@rci.rutgers.edu
Daniel Gonzalez
Rutgers University, Cook College
Biochemistry and Microbiology
76 Lipman Drive
New Brunswick, NJ 08901-8525
Daniel thanks Turner BioSystems for making
this application note possible and for providing the GFP community with
a useful analysis tool in their TD-700 fluorometer. |
