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.

Gene Activation and Cloning:The BBa_K592100 construct was activated from the distribution kit and the mTagBFP gene was homologously recombined into the pET29a(+) 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 hours. Since the plasmid is expressed in the pET29a(+) 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 competent E. coli BL21(DE3) strain. After incubating in 5 mL LB medium for 12 hours, the culture was scaled up by transferring it into 50 mL LB medium and incubated until the optical density (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 hours. 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, vary the excitation wavelength to measure the fluorescence emission intensity of the samples in order to determine the optimal excitation wavelength for mTagBFP. Subsequently, fix the excitation wavelength and measure 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) cells 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, across a temperature range of 25°C to 90°C. Fluorescence changes were continuously monitored as the temperature gradually increased during 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.

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.

Using the fluorescence signal-to-noise ratio formula SNR=Inoise/Isignal, it can be observed that under excitation at 401 nm, the signal-to-noise ratio of the mTagBFP blue fluorescent protein emission in E. coli BL21(DE3) steadily increases from around 420 nm to 450 nm, reaching a peak of 3.48 at 453 nm, after which it slowly declines while remaining at a relatively high level.

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°C to 90°C, with a heating rate of 0.5°C/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.

Based on the test results, the average Tm value of the blue fluorescent protein mTagBFP is 57.32°C(±2.68°C).