Protocol for Assaying 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 mm 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)
• 13mm x 100mm 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.

1. Follow the procedure outlined in your operation manual to install the near UV mercury vapor lamp.
2. 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.
3. 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

1. Close the TD-700 lid and turn the unit on. It will count down 600 seconds to warm up.
2. 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.
3. 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.
4. Enter [3] to enter the units menu, then use the ARROW key to select µg/mL. Press [enter] to return to the setup menu.
5. Enter [2] to enter the calibration procedure menu, and select DIRECT CONCENTRATION using the ARROW key. Press [enter], then [ESC].
6. 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.
7. 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.
8. 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.
9. 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

1. 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.
2. 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).
3. Perform the above mentioned calibration and quantitation procedure (sections 3 and 4) to estimate the concentration of GFP in your transformed cells.
4. 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

1. A suggested method for the quantitation of green fluorescent Protein. Turner BioSystems Application note. 1996.
2. 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.