RecA(BBa_K3020001)-EGFP and RecA(BBa_K629001)-EGFP

---

RecA(BBa_K629001)-EGFP (BBa_K4814014)

RecA(BBa_K3020001)-EGFP (BBa_K4814015)

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 correlation between SFU 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 performance of the BBa_K3020001 promoter was superior to that of the BBa_K629001 promoter, indicating that the optimized BBa_K3020001 promoter was able to reduce background noise.

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 1. 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 2. The fluorescence of RecA(BBa_K3020001)-EGFP, RecA(BBa_K629001)-EGFP with different concentrations of Aspartame and Nalidixic 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.

As depicted in Figure 3, 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.

The imaging results (Fig. 4) 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.

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.

RBS test of B0032 (BBa_K4814002), Strong (BBa_K4814013), and B0034 (BBa_K4814015)

---

Confirming the excellence of BBa_K3020001 reporter, we aimed to further improve the reporter by replacing RBS B0034 (our original design) with RBS B0032 and new RBS strong.

---

The following charts are the SFU (significant fluorescence units. fl over OD600) of DNA damage reporter with different RBS (Strong BBa_K4814013, B0032 BBa_K4814002, and B0034 BBa_K4814015). The RecA-EGFP design with B0034 RBS is the original design (BBa_K3020001). We replaced B0034 with B0032 and a new RBS generated by machine learning results by Zhang, M. et al (2022). This experiment aims to select the best design by comparing the fluorescence over OD600 when exposed to genotoxic agents (UV, H₂O₂, Aspartame, and Nalixidic acid).

Figure 1. The graphs of different RBS designs treated with UV, H₂O₂, Aspartame, and Nalidixic acid.

We calculated the Standard Error (SE) with n = 3:

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

Overall, the SFU of all designs has an upward trend in UV and H₂O₂ groups. The results illustrate that while in both UV and H₂O₂ groups, the SFU of B0032 (BBa_K4814002) is three times of B0034 (BBa_K4814015), the Strong (BBa_K4814013) is only half of B0034.

However, when it comes to Aspartame, the SFU of all engineered bacteria decreases when the concentration of Aspartame increases. On the other hand, the SFU of Strong and B0034 rises from 0 to 10 ug/ml of Nalidixic acid, followed by a steep drop between 10 and 100.

Hha biofilm reducer

---

The hha protein BBa_K4814001 is encoded to reduce biofilm formation and is designed to target and kill bacteria upon activation of the RecA promoter in response to DNA damage. In this study, our objective was to examine the impact of increasing concentrations of carcinogens on the optical density (OD) of a bacterial culture.

Initially, we hypothesized that as the concentration of carcinogens increased, the OD would decrease due to the bactericidal effects of the activated hha protein. However, the experimental results revealed that despite the escalating level of carcinogen treatment, the OD did not exhibit a significant decrease compared to the control group (top10 or E. coli), nor did it demonstrate a discernible trend. This could be attributed to adaptive responses to stress or incomplete protein folding of the hha protein.

As illustrated in Figure 1, the OD of the TOP10 control group decreased slightly from 0.28 to 0.26, and then increased to 0.3 after 6, 12, and 18 minutes of UVB exposure. In contrast, starting at 0.24, the OD of the hha group showed a slight increase and then decreased to 0.22 after 18 minutes of UVB treatment. In the graph representing the H₂O₂ group (Figure 2), both the hha and TOP10 groups exhibited a decreasing trend in OD, consistent with our initial hypothesis.

To further investigate this phenomenon, future studies can assess the expression level of the hha protein using techniques such as Western Blot or qPCR. These analyses will provide insights into the protein's expression patterns and help elucidate the underlying mechanisms contributing to the observed OD changes.

Figure 1 & 2. The graph of the OD600 of RecA(BBa_K629001)-hha after being treated with UVB (left) and H₂O₂ (right).

Figure 3 & 4. The graph of the OD600 of RecA(BBa_K629001)-hha after being treated with Nalidixic acid (left) and Aspartame (right).

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

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

ATRIP-EGFP UV treatment

---

ATRIP and RPA1 are crucial proteins involved in DNA damage signaling pathways within human cells. Once the RPA protein binds to single-stranded DNA, it recruits the ATR complex by interacting with the ATRIP protein. To investigate this interaction and detect DNA damage events in living human cells, we employed a fluorescence resonance energy transfer (FRET) approach by fusing two fluorescent proteins, namely EGFP & mCherry and ECFP & EYFP. This allowed us to measure the rate of energy transfer and determine the ATRIP-RPA1 interaction.

The following designs were utilized in our study:

• ATRIP-EGFP (BBa_K4814006)
• RPA1-mCherry (BBa_K4814007)
• ATRIP-ECFP (BBa_K4814008)
• RPA1-EYFP (BBa_K4814009)

Aggregation after UV treatment

---

After exposing the cells to a UVB dosage of 100 J/m^2, we observed aggregation of the EGFP signal (Fig. 1 and 2). Interestingly, fluorescence was detected in both the Green and Red channels. It is important to note that the emission of GFP is dependent on its fluorescence spectra, as mentioned in studies by Sattarzadeh, A. et al. (2015) and Licea-Rodriguez, J. (2019). This fluorescence could potentially be attributed to GFP emitting at around 560 nm.

Figure 1 (top) & 2 (bottom). The image of ATRIP-EGFP after UVB 100 J/m^2 exposure. Both tests showed clusters and aggregation of signal in green channel and red channel. (488 nm excitation)

FRET (ATRIP-EGFP + RPA1-mCherry)

---

After subjecting the cells to UVB treatment at a dosage of 100 J/m^2, we observed a change in the density of both EGFP and mCherry signals. When excited at 488 nm, we noticed that the EGFP signal became weaker following exposure to UVB. However, in contrast, the red fluorescence emitted by mCherry (with an emission range of 570-620 nm) intensified.

Figure 1. The image of ATRIP-EGFP (excited at 488 nm) + RPA1-mCherry (RPA1-mCherry transfected twice) (excited at 561 nm).

To enhance the accuracy and reliability of our data analysis, we utilized ImageJ software to precisely outline the cell nuclei present in the Green Channel (excited at 488 nm). This step ensured that we specifically selected cells that were transfected with EGFP, as depicted in Figure . We focused on GFP-emitting cells because we observed that the image captured in the 488 nm excited green channel did not completely overlap with the image in the 488 nm excited red channel. However, it is important to note that the 488 nm red channel fluorescence should correspond to GFP emission at approximately 560 nm. Therefore, the shape of the cells in the red channel should be identical to that in the green channel (as shown in Figure .). By choosing GFP-emitting cells, we aimed to reduce background noise and focus our analysis specifically on the G+M cells (cells expressing both ATRIP-EGFP and RPA1-mCherry), excluding cells expressing only RPA1-mCherry.

Figure 2. The image of green channel (left) and circled merged channel (green and red) of G+M excited at 488 nm.

Statistical Analysis

---

We calculated the ratio of FRET using the Red over Green (Channel 3 over Channel 2) ratio. When the ratio is bigger than 1, there is more red fluorescence in the cell. We can compare the ratio before and after UV treatment to determine whether FRET occurs.

To handle the non-linear nature of the data, we took the logarithm of the values with a base of 2, which brings the values onto a comparable scale.

In Figure 3, the data points in the UV- graph are divided into two groups, with a separation occurring at 0.1. We set this value as the cutoff point, indicating the presence of FRET when the data point exceeds 0.1.

In the UV+ graph, there is a noticeable distinction in the proportion of data points indicating FRET.

Figure 3. Distribution of the Log base 2 Red/Green data before and after UV.

The Red over Green ratio (Log_2) showed an increase of more than threefold after UVB light treatment. This substantial increase indicates that there is energy transfer from GFP to mCherry, resulting in the emission of red fluorescence when exposed to UV light. This confirms the occurrence of FRET energy transfer.

To assess the significance of the relationship between the two categorical variables, we employed Fisher's exact test. This statistical test is suitable when dealing with small cell counts. When the two-sided p-value is less than 0.01, it suggests a significant association between the two groups. (MedCalc Software Ltd. Fisher, 2023)

The result of Fisher's exact test revealed a strong significance between the two groups (p-value = 0.00122178, p-value < 0.01), indicating a stastical significance.

The calculator we used:

https://www.medcalc.org/calc/fisher.php

Figure 15. Mean value (with error bar, technical sample number = 4, about 30 data points in each sample) of logarithms of the data with base 2 before and after UV.

Figure 16. Fisher's exact test result: p value < 0.05.