Measurement

The goal of our project was to develop a biosensor utilizing a split GFP System. That would allow us to monitor transcriptional activity of a gene cluster in vivo. We originally planned on relying on the strenght of the GFP fluorescence to be strong enough to be visible to the naked eye on LB-Agar plates. But after plating our first E. coli Dh5a pNit_sfGFP we very quickly noticed that you could barely tell a difference betwen GFP and non-GFP colonies. Because of this we set out to develop a method that would allow us to reliably and quickly detect even faint fluorescence signals. For this we tested:

  • Fluorescence microscopy
  • Fluorescence measurement using a platereader

Intro Image

Fluorescence microscopy

We first used fluorescence microscopy to detect sfGFP fluorescence signals. With the assistance of Dr. Christine Keimer, we used an Olympus BX51 microscope equipped with a 100x objective, a CCD camera (Retiga 3; QImaging), and an LED light source (Sola 365; Lumencor) for imaging. The excitation filter was set for 450-490 nm and the emission filter for 500-550 nm to image E. coli DH5α pNit_QC1_sfGFP and E. coli DH5α pNit_QC1 (non-GFP control).[1] We observed a clear difference in fluorescence intensity between the sfGFP sample and the non-GFP control. However, Dr. Keimer explained that this setup cannot be used to quantify fluorescence intensity due to auto-scaling. Additionally, the need to schedule microscope time for each measurement led us to explore an alternative method using a plate reader.

Figure 1 E. coli Dh5a pNit_QC1_sfGFP (left) and E. coli Dh5a pNit_QC1 (right) under the fluorescence microscope.

Fluorescence measurement using a platereader

Due to the fact that LB-medium cotains a lot of organic compounds it causes a lot of background fluorescence because of this we harvested our cells via centrifugation discarded the supernatant and resuspended the cells in dH2O. 150 µl of the resuspended cells were then transferred to a black 96 well plate and measured in a platereader (TECAN Infinite 200 PRO). The excitation wavelength was set to 488 nm and the emission wavelength to 514 nm. We were able to detect a significant increase in fluorescence intensity in the sfGFP samples compared to the non-GFP control samples.

Figure 2 Fluorescence intensity of E. coli Dh5a pNit_QC1_sfGFP (blue) and E. coli Dh5a pNit_QC1 (orange) measured with a platereader, excitation wavelength 488 nm, emission wavelength 514 nm.

Conclusion

Although we were unable to establish a functional split GFP system, we successfully demonstrated that both measurement methods could detect sfGFP fluorescence signals. During sample preparation for plate reader measurements, we washed the samples to replace the LB medium with distilled water (dH₂O) in order to reduce background fluorescence. While harvesting, we observed that the cell pellets from the pNit_sfGFP samples were visibly greener than those from the non-GFP control samples. While unintended, this observation suggests that concentrating cells via centrifugation to enhance fluorescence visibility could serve as a simple, rapid screening method to confirm successful protein expression, requiring no additional equipment beyond a basic centrifuge.

Figure 3 Cell pellets of E. coli Dh5a pNit_QC1_sfGFP (left) and E. coli Dh5a pNit_QC1 (right) after centrifugation.

References

[1] Thiery, S.; Turowski, P.; Berleman, J. E.; Kaimer, C. The Predatory Soil Bacterium Myxococcus Xanthus Combines a Tad- and an Atypical Type 3-like Protein Secretion System to Kill Bacterial Cells. Cell Rep. 2022, 40 (11), 111340. https://doi.org/10.1016/j.celrep.2022.111340.