For the contribution part of our project, we undertook a systematic characterization of the fluorescence signal-to-noise ratio and thermal stability of the blue fluorescent protein mTagBFP. As a widely utilized fluorescent marker, mTagBFP is favored for its rapid maturation and distinct spectral properties. However, its excitation and emission wavelengths fall within the near-UV region, making it susceptible to background fluorescence interference. Accurately measuring its signal-to-noise ratio is therefore critical for obtaining high-quality data, particularly in complex backgrounds. Additionally, the thermal stability of mTagBFP directly impacts its fluorescence performance under varying temperature conditions. Despite its importance, no research team has yet systematically characterized mTagBFP when expressed in prokaryotic systems.

We inserted the BBa_K592100 part into the pET-29a(+) vector, and then transformed the recombinant plasmid into E. coli DH5α for amplification, followed by transformation into BL21(DE3) for protein expression. We measured the fluorescence signal-to-noise ratio and thermal stability of mTagBFP both in vivo within E. coli and in vitro. These data will be invaluable for subsequent research teams, enabling them to optimize experimental conditions, reduce the impact of background interference and temperature fluctuations on signal stability, and ultimately enhance the reliability of their experimental outcomes.

Fig 1 | The Plasmid Map of pET-29a(+)-mTagBFP

Gene Activation and Cloning: The BBa_K592100 construct was activated from the distribution kit and the mTagBFP gene was homologously recombined into the pET-29a(+) vector. This recombinant vector was then transformed into E. coli DH5α strain, which were cultured overnight at 37°C. Two single colonies were selected and inoculated into 5 mL of LB medium for 12 h. Since the plasmid was expressed in the pET-29a(+) vector, both LB agar plates and media were supplemented with Kanamycin at 50 μg/mL.

Plasmid Extraction and Protein Expression: The plasmid was extracted and transformed into E. coli BL21(DE3). After growing in 5 mL LB medium at 37°C for 12 h, the bacteria culture was transferred into 50 mL LB medium until the optical density at 600 nm (OD₆₀₀) reached 0.6-0.8. IPTG was then added to induce protein expression, and the culture was further incubated at 16°C for 20 h. Protein extraction was performed using an ultrasonic cell disruptor.

Preliminary Fluorescence Measurements: A pre-experiment was conducted using a microplate reader to determine the excitation and emission wavelengths of the expressed blue fluorescent protein.

We transferred 200 μL of bacterial culture or protein sample into a black opaque 96-well plate. Using a PerkinElmer Ensight multimode plate reader, we varied the excitation wavelength to measure the fluorescence emission intensity of the samples in order to determine the optimal excitation wavelength for mTagBFP. Subsequently, we fixed the excitation wavelength and measured the emission spectra of the samples.

Fluorescence Signal-to-Noise Ratio Measurement: Based on preliminary results, we quantified the fluorescence intensity of the blue fluorescent protein mTagBFP expressed in E. coli BL21(DE3) and the background fluorescence of the E. coli BL21(DE3) cells themselves. The signal-to-noise ratio of mTagBFP expressed in E. coli BL21(DE3) were then calculated and evaluated.

Thermal Stability Characterization: Building on preliminary findings, we used the Thermo Fisher QuantStudio^™ 5 real-time PCR system to assess the thermal stability of mTagBFP both in vivo and in vitro over a temperature range from 25°C to 90°C. Fluorescence changes were continuously monitored as the temperature gradually increased throughout the experiment.

Based on our characterization, the excitation wavelength of mTagBFP blue fluorescent protein in E. coli BL21(DE3) is 401 nm and the emission wavelength is 474 nm. Under excitation at 401 nm, the emission in the 450 nm-480 nm range is significantly more pronounced compared to background fluorescence, making it more easily distinguishable as a reporter signal.

Fig 2 | Excitation/emission wavelength of mTagBFP in E. coli  BL21(DE3).

In vitro, the excitation wavelength is 403 nm and the emission wavelength is 471 nm. The emission intensity in the 450 nm-485 nm range is relatively high and stable.

Fig 3 | Excitation/emission wavelength of mTagBFP in vitro

Using the fluorescence signal-to-noise ratio formula SNR = I_noise / I_signal, we observed that the signal-to-noise ratio of mTagBFP emission in E. coli BL21(DE3) increased steadily under excitation at 401 nm. The SNR rose from approximately 420 nm to 450 nm, reaching a peak of 3.48 at 453 nm. After this point, it slowly declined but remained at a relatively high level.

Fig 4 | The emission signal-to-noise ratio of mTagBFP in E. coli  BL21(DE3)

We assessed the thermal stability of the mTagBFP blue fluorescent protein using the Thermo Fisher QuantStudio^™ 5 real-time PCR system. We added 20 μL of bacterial culture or protein sample to each well of a 96-well plate. The experiment was conducted with a temperature range from 25℃ to 90℃, with a heating rate of 0.5℃/s. The melting curve of mTagBFP was measured to ensure a comprehensive evaluation of the protein's thermal stability. Fluorescence changes were continuously monitored as the temperature gradually increased during the experiment.

Fig 5 | The melting curve mTagBFP in vivo

By analyzing the melting curve of the mTagBFP blue fluorescent protein, we obtained its average melting temperature (Tm) value of 57.32℃(±2.68℃). This Tm value reflects the temperature at which the protein begins to significantly unfold during heating, leading to its inactivation, and provides an important indicator of the thermal stability of mTagBFP.