ATRIP-EGFP and RPA1-mCherry/ ATRIP-ECFP and RPA1-EYFP FRET

HEK293 ATRIP and RPA1 localization

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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)

To initially assess the localization of ATRIP and RPA1 and confirm the successful transfection of our desired vectors, we conducted imaging experiments of fluorescent protein (EGFP, mCherry, ECFP, and EYFP) tagged proteins in HEK293 cells.

The choice of HEK293 cell line was based on several factors, as supported by previous studies (Thomas, P., & Smart, T. G., 2005; ThermoFisher, n.d.):

1. Its high capacity for producing recombinant proteins;
2. Its efficient post-translational folding and processing machinery;
3. Its capability of expressing proteins from both mammalian and non-mammalian nucleic acids;
4. HEK293 cells are easily reproducible and maintainable;
5. Its amenity to transfection using various methods, thus facilitating experimental manipulations.

Vector transfection

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Following transfection, we performed selection using hygromycin (HygR) and puromycin at concentrations of 500 μg/ml and 0.8 μg/ml, respectively. Subsequently, the cells were transferred to confocal dishes with a diameter of 35 mm.

To visualize the nucleus, we utilized Hoechst blue staining. As depicted in the graph, both ATRIP-EGFP and RPA1-EYFP displayed distinct localization patterns that completely overlapped with the nucleus. However, the signal from mCherry was indistinct, exhibiting a similar intensity to the background signal. (Fig. 1) In comparison to the control HEK293 cells (Fig. 2), the red fluorescence of mCherry, excited at 561 nm, was considerably low.

Figure 1 & 2. The localization of ATRIP-EGFP, RPA1-mCherry, RPA1-EYFP, and ATRIP-EGFP + RPA1-mCherry (left). The HEK293 cells imaging before and after UV treatment (right).

In addition, we conducted transfections with ATRIP-ECFP and ATRIP-ECFP + RPA1-YFP (C+Y). However, due to time constraints images of the cells were taken in the six-well plate and the quality was slightly compromised. Nonetheless, these images still provide confirmation that both vectors were successfully transfected into the cells. The CFP fluorescence was excited at 405 nm and the YFP fluorescence was excited at 488 nm. (Fig. 3)

Figure 3. The images of ATRIP-ECFP and ATRIP-ECFP + RPA1-EYFP (C+Y) on six-well plate.

Double Transfection of RPA1-mCherry to ATRIP-EGFP + RPA1-mCherry (G+M)

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As a strong mCherry signal was not observed, we hypothesized that the vector might not have been effectively transfected into the cells. Another potential explanation could be that the emission wavelength channel we selected was unable to detect the mCherry signal. To address this issue, we packaged the RPA1-mCherry vector into a virus and performed a subsequent infection in cells already containing ATRIP-EGFP and RPA1-mCherry (G+M). Figure 4. depicts the improved image of ATRIP-EGFP + RPA1-mCherry (G+M, mCherry twice infected) following this approach.

Figure 4. The image of ATRIP-EGFP (excited at 488 nm) + RPA1-mCherry (RPA1-mCherry transfected twice) (excited at 561 nm).

Aggregation after UV treatment

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Next, we subjected the HEK293 cells expressing ATRIP-EGFP to UV treatment in order to test the localisation of the ATRIP protein. According to Wu, C. S. et al. (2014), the ATRIP protein is expected to aggregate upon UV treatment. Our hypothesis was that after UV exposure, ATRIP-EGFP would concentrate in the nucleus, forming distinct clusters.

Before initiating the treatment, we captured images of the cells. Subsequently, we exposed the cells to UVB light within the range of 280 nm to 315 nm, applying a dosage of 10 J/m^2 as per the conditions described by Wu, C. S. and their team. After an hour of incubation, the cells were photographed again.

Furthermore, the mCherry signal was observed to be very weak, almost indistinguishable from the background. When compared to the control group (HEK293 cells), it is evident that the transfection of RPA1-mCherry was not successful. (Fig. 6)

Despite our hypothesis, the GFP signal did not aggregate as expected. (Fig. 5) We suspect that this might be due to the relatively short incubation period of only one hour.

Figure 5. ATRIP-EGFP image after treating with UVB.

Likewise, following the UV treatment, the mCherry signal (in singly transfected cells) exhibited no significant changes. (Fig. 6) Additionally, the mCherry signal remained at roughly the same level as the background. Comparing it with the control group (HEK293 cells), it is evident that the successful transfection of RPA1-mCherry did not occur. Please note that we have not specifically tested the UV treatment of RPA1-mCherry alone, but rather the combination of ATRIP-EGFP and RPA1-mCherry.

Figure 6. RPA1-mCherry image after treating with UVB.

In the case of RPA1-EYFP, red fluorescence emerged after UV treatment. (Fig. 7)

Figure 7. RPA1-EYFP image after treating with UVB.

In an effort to improve our previous results, we extended the incubation time to two hours. (Fig. 8) However, the GFP signal still did not aggregate to the extent we anticipated. As shown in the graph, the GFP appeared to concentrate in certain regions of the cells, but the aggregation was not significant or widespread.

During our investigation, we came across research by Ravanat, J.-L., Douki, T., & Cadet, J. (2001), which highlighted that most previous studies utilized UVC lamps (254 nm) for cell treatment. However, this wavelength is not biologically relevant for sun exposure. Additionally, UVB and UVA light at doses below 25 J/m^2 had minimal effects on the cells, as stated in the study conducted by Kimura, H., et al. (2010). Furthermore, it is worth noting that UVB light starts exhibiting cytotoxic effects on cancer cells after a dosage of 50 J/m^2. Therefore, in future experiments, we should consider using higher doses of UVB to achieve the desired outcomes.

Figure 8. The ATRIP-EGFP image after UV treatment.

Finally, after exposing the cells to a UVB dosage of 100 J/m^2, we observed aggregation of the EGFP signal. (Fig. 10 & 11) 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). (Fig. 9) This fluorescence could potentially be attributed to GFP emitting at around 560 nm.

Figure 9. The absorption and emission spectrum of CFP, GFP and RFP. Obtained from: Semrock, Inc., Semrock Catalog, 2018, www.semrock.com (2018).

Figure 10 & 11. 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)

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

Selecting the ATRIP-EGFP expressing cells

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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 13. We focused on GFP-emitting cells because the number of cells in the 488 nm excited green channel is not the same as those in the 488 nm excited red channel. It is important to note that the 488 nm red channel fluorescence should correspond to GFP emission at approximately 560 nm. Therefore, the shape and number of the cells in the red channel should be identical to that in the green channel (as shown in Figure 10 & 11.).

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

Statistical Analysis

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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 14, 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 (value > 0.1).

Figure 14. 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 UV- and UV+ 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 < 0.01.

Hha biofilm reducer

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The hha protein BBa_K4814000 is encoded to reduce biofilm formation and is designed (BBa_K4814001 composite part) 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(K6)-hha after being treated with UVB (left) and H₂O₂ (right).

Figure 3 & 4. The graph of the OD600 of RecA(K6)-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.