RecA(BBa_K3020001)-EGFP test with UV, H2O2, Nalidixic acid, and Aspartame


In order to validate the findings of a previous iGEM team and investigate the genotoxic effects of carcinogens, we conducted dosage-dependent tests on the TOP10 E. coli. We utilized the RecA (BBa_K3020001) promoter by BIT 2019 in conjunction with EGFP (BBa_K3020002) as a bioreporter to detect DNA-damaging agents such as UV, H2O2 and nalidixic acid. This promoter is an optimized version of BBa_K629001 (K6), a design by team SYSU 2011 aimed at driving the motor system of E. coli in a radioactive environment.

To determine the comparative DNA-damage detection ability of the RecA(BBa_K3020001) and RecA(BBa_K629001) promoters, we designed RecA(BBa_K3020001)-EGFPBBa_K4814015 and RecA(BBa_K629001)-EGFP BBa_K4814014constructs, and measured the GFP emission from these two promoter designs when exposed to carcinogens.

Initially, the bacteria were treated with carcinogens and transferred into 96-well plates to measure fluorescence and optical density (absorbance at 600nm). The dosage references were obtained from team BIT 2019. Additionally, we tested Aspartame at a concentration of 1 g/L based on the study by Yılmaz and Uçar (2014).

To compare the EGFP signal among different groups and analyze the fluorescence relative to cell growth (OD600), we utilized the fluorescence over OD600 (FL over OD) ratio. Following the methodology described by team BIT 2019 (https://2019.igem.org/Team:BIT/Bio), we used specific fluorescence units (SFU = RFU/OD600) to quantify fluorescence. After comparing the data from three independent experiments, we observed a direct positive correlation between EGFP fluorescence intensity and the intensity/concentration of the carcinogens, specifically UV and H2O2. In the aspartame group, both BBa_K3020001 and BBa_K629001 exhibit a marginal increase in specific fluorescence units (SFU). This phenomenon can be attributed to the complex and indirect mechanism by which aspartame may potentially contribute to cancer development. Moreover, the fluorescnece of the BBa_K3020001 promoter was greater than that of the BBa_K629001 promoter, indicating that the optimized BBa_K3020001 promoter was more sensitive and able to reduce background noise.

Figure 1. The fluorescence of RecA(BBa_K3020001)-EGFP, RecA(K6)-EGFP with different intensity of UVB and concentrations of H2O2. Data is collected three hours after the treatment.
Figure 2. The fluorescence of RecA(BBa_K3020001)-EGFP, RecA(BBa_K629001)-EGFP with different concentrations of Aspartame and Nalixidic acid. Data is collected three hours after the treatment.

We calculated the standard error (SE), with n=3:

where σ is the standard deviation, and n is the number of trials.

We sought guidance from our advisors, Prof. Leo Tsz On Lee and Prof. Tzu-Ming Liu from the University of Macau, to obtain a more robust approach for quantifying the fluorescence emission of each bacterium. They recommended employing cell imaging techniques utilizing microscopes.

Upon further investigation, we observed that bacteria containing the RecA-EGFP plasmid exhibited green fluorescence even without any treatment. Consequently, to obtain accurate and objective measurements of the GFP levels, we employed image processing software such as ImageJ. This enabled us to analyze the images and derive reliable results rather than relying on subjective assessments to determine the GFP quantities.

Figure 3. The gel with bacteria is placed on the slide and covered with coverslip.
Figure 4. The bright field (left) and GFP channel image (right) of RecA(BBa_K629001)-EGFP E.coli has no treatment.

It is evident that the disparity between group (c) and (d) (RecA K6 without H2O2 and with H2O2) is not highly significant. However, individual bacteria within group (b) (RecA(BBa_K3020001)-EGFP treated with H2O2) exhibit noticeably higher fluorescence intensity compared to group (a) (no treatment). Nevertheless, due to the absence of a readily discernible difference, we opted to employ ImageJ software for calculating the mean values. (August 8th)

RecA(BBa_K3020001)-EGFP no treatment RecA(BBa_K3020001)-EGFP H2O2 5mM
RecA(BBa_K629001)-EGFP no treatment RecA(BBa_K629001)-EGFP H2O2 5mM
Figure 5. (a) RecA(BBa_K3020001)-EGFP group without treatment, bright field and GFP channel images. (b) RecA(BBa_K3020001)-EGFP group treated with H2O2 5mM. (c) RecA(BBa_K629001)-EGFP group without treatment. (d) RecA(BBa_K629001)-EGFP group treated with H2O2 5 mM.

In certain instances, E. coli tends to aggregate and form clusters of fluorescent units. To ensure accurate analysis, we exclusively considered objects whose area ranged from 100 to 3000 square units. By restricting the selection to this specific range, we generated box plots using the resulting data points. The raw images were captured using a 100x confocal microscope, utilizing the GFP channel (488). Subsequently, the images were processed using ImageJ, applying an 8-bit format and RenyiEntropy threshold auto-adjustment.

Results

The images were taken with a confocal microscope using an EGFP channel, exposure time of 80.024 ms. As depicted in Figure 6, the fluorescence intensity of RecA(BBa_K3020001)-EGFP bacteria exhibits an upward trend with increasing duration of UVB exposure. This observation suggests that UVB radiation induces the SOS response in bacteria. A similar pattern is observed in the other treatment groups, including H2O2, nalidixic acid, and aspartame. Through this experiment, we have successfully validated the efficacy of utilizing the RecA promoter and EGFP fluorescent protein combination for evaluating genotoxicity based on the extent of DNA damage.

Figure 6. The box chart using analysis of imaging results: (a) The graph demonstrates the fluorescent units of bacteria treated with UVB at intensity 20 for 6, 12, and 18 minutes. All photos were taken 2-3 hours after the treatment. (b) Fluorescence of cells treated with H2O2 at 0.05, 0.5, 5 mM. (c) Fluorescence of cells treated with nalidixic acid at 1, 10, and 100 μg/ml concentration. (d) Fluorescence of cells treated with Aspartame at 0.25, 0.5, and 1 μg/ml concentration.

A dot = the mean fluorescence value of a bacteria. Independent sample number = 3; at least 30 cells in each sample.

All figures above have one-way ANOVA significance p < 0.001, with significance comparison (with No Treatment) indicated by: ns = no significance; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

RecA(BBa_K629001)-EGFP test with UV, H2O2, Nalidixic acid, and Aspartame


The data demonstrates the RecA(BBa_K629001)-EGFP construct’s inferior performance in reporting DNA damage compared to RecA(BBa_K3020001)-EGFP across all treatment groups (see Fig. 1-2). Notably, in the aspartame group, the SFU of RecA(BBa_K3020001)-EGFP is over three times higher than that of RecA(BBa_K629001)-EGFP.

Figure 7. The fluorescence of RecA(BBa_K3020001)-EGFP, RecA(BBa_K629001)-EGFP with different intensity of UVB and concentrations of H2O2. Data is collected three hours after the treatment.
Figure 8. The fluorescence of RecA(BBa_K3020001)-EGFP, RecA(BBa_K629001)-EGFP with different concentrations of Aspartame and Nalixidic acid. Data is collected three hours after the treatment.

The images were taken with a confocal microscope using an EGFP channel, exposure time of 80.024 ms. The box chart using analysis of imaging results (Fig. 9) of the BBa_K629001 group (E. coli with RecA(BBa_K629001)-EGFP) indicate a lack of significant dosage-dependent response. This can be attributed to the enhanced performance of RecA(BBa_K3020001)-EGFP, which has undergone sequence optimization. The fluorescence intensity in the UVB exposure groups ranging from 6 to 18 minutes remained relatively unchanged. Similarly, in the aspartame and nalidixic acid groups, the fluorescence exhibited minimal variations. Conversely, the H2O2 group displayed a slight decrease, but the overall changes were not significant.

Figure 9. RecA(BBa_K629001)-EGFP group: (a) The graph demonstrates the fluorescent units of cells treated with UVB at intensity 20 for 6, 12, and 18 minutes. All photos were taken 2-3 hours after the treatment. (b) Fluorescence of cells treated with H2O2 at 0.05, 0.5, 5 mM. (c) Fluorescence of cells treated with nalidixic acid at 1, 10, and 100 ug/ml concentration. (d) Fluorescence of cells treated with Aspartame at 0.25, 0.5, and 1 g/ml concentration.

A dot = the mean fluorescence value of a bacteria. Independent sample number = 3; at least 30 cells in each sample.

All figures above have one-way ANOVA significance p < 0.001, with significance comparison (with No Treatment) indicated by: ns = no significance; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

References

Yılmaz, S., & Uçar, A. (2014). A review of the genotoxic and carcinogenic effects of aspartame: does it safe or not?. Cytotechnology, 66(6), 875–881. Yılmaz, S., & Uçar, A. (2014). A review of the genotoxic and carcinogenic effects of aspartame: does it safe or not?. Cytotechnology, 66(6), 875–881. https://doi.org/10.1007/s10616-013-9681-0