Introduction
The worldwide cancer crisis is an urgent issue that demands immediate attention. As one of the leading causes of death globally, cancer continues to affect millions of lives each year. Its devastating impact lies in the profound toll it takes on human health, well-being, and economic stability. Meanwhile, the cancer epidemic is becoming more prevalent, and communities that were previously relatively unharmed are starting to experience the wrath of this disease. Over the past several decades, the incidence of early-onset cancers has increased in multiple countries (Ugai et al., 2022).
Macau, where we are living, is a vibrant urban city with a fast-paced lifestyle that also faces the same challenge due to the presence of carcinogens in everyday life. The modern diet and products used by the population are often exposed to these harmful substances. Common carcinogens include phthalates in personal care products[1], heavy metals in cosmetics (Borowska & Brzóska, 2015), and nitrosamines and PAHs in certain foods like processed meats and charred foods (Carpenter & Bushkin-Bedient, 2013). Unfortunately, these carcinogens are pervasive, resulting in Macau experiencing a high prevalence of cancer: ranking 48th worldwide for cancer incidence (Cancer Rates by Country 2023, n.d.), and showing a steady increase (ANNUAL REPORT OF MACAO CANCER REGISTRY, 2020) (Fig. 1). While screening programs, such as the “Lung Cancer Screening Pilot Program” have been implemented to improve this situation in recent years, extra input has to take place to reduce the cancer incident rate.
Limitations of Carcinogenic Toxicity Screening
Given the unavoidable and increasing use of various chemical applications in our daily products, we have decided to undertake the challenge of reducing the exposure of potential carcinogenic toxicity in our society by detecting carcinogenic toxicity. Currently, cell-based assays have been employed to detect toxicity in different scenarios, however, they come with limitations. The crisis of the global manufacturing chain and the food supply highlights the potential unmet demand for chemical reagents, taking into account resource allocation. As reagent production is foreseen not to meet future demands, this situation can hinder widespread application in toxicity screening. Secondly, current DNA damage detection methods need more sensitivity and specificity (Collins, 2004), (Nikitaki et al., 2015), such as the inability to detect low levels of DNA damage or differentiate between different types of DNA damage. Moreover, some methods can be time-consuming (Van Dierendonck, 2003), require large amounts of DNA, and may not be suitable for high-throughput analysis. These limitations highlight the need for the development of more sensitive, specific, and reliable DNA damage detection methods. Therefore, we are proposing the establishment of our reporter line system.
A Potential Solution: Biosensors
Previous literature proves that specific genes are activated in response to toxic and oxidative drugs and chemicals (Podlesek & Bertok, 2020), (Ball et al., 2005); while under normal conditions, the expression of which is limited. Here, we propose to tackle the issue above by developing innovative DNA-damage bioreporter systems based on these DNA-damage-sensitive genes. Through discussing and analyzing DNA damage genes and others with specific functions, we have selected our target genes for developing our toxicity screening platform. Our proposed platform is based on reporter cell lines (human and E. coli). Our proposed methods allow DNA damage to be observable without chemical reagents, allowing for efficient and environmentally conscious quantifying carcinogenic toxicity. In addition, such innovative development has the potential to revolutionize toxicity testing, providing valuable insights into toxicity-induced DNA damage while minimizing the environmental impact associated with traditional methods.
RecA-EGFP reporter system
Firstly, we validated the RecA promoter, designed by team SYSU in 2011 as BBa_K629001 (K6) and improved by team BIT in 2019 as BBa_K3020001 (BBa_K3020001). When single-strand breaks are induced in E. coli, the SOS response takes place, in which RecA binds to the damaged DNA and converts to an activated form (RecA nucleoprotein filament). Its activation stimulates the self-cleavage of the LexA dimer, a transcriptional repressor, lowering its affinity for the DNA and thereby deactivating it. As the pools of LexA protein begin to decrease, the RecA promoter is activated and its downstream genes are derepressed. (Masłowska et al., 2019)
SYSU showed that this promoter is activated when E. coli is treated with UV, and BIT2019 optimized the promoter sequence to reduce background fluorescence when detecting single-strand breaks. Our team initially designed RecA(BBa_K3020001)-B0034-EGFP and RecA(BBa_K629001)-B0034-EGFP to measure the intensity of GFP emission after different degrees of DNA-damage-inducing treatment, thereby validating and distinguishing the performances of BBa_K3020001 and BBa_K629001.
Our team showed that the promoter is activated when treated with different intensities of UV and concentrations of hydrogen peroxide, aspartame, and nalidixic acid. Moreover, the performance of the BBa_K3020001 promoter was superior to that of the BBa_K629001 promoter, validating that the optimized BBa_K3020001 promoter was indeed more sensitive and able to reduce background noise. Having its superior performance, we sought to further improve our construct by designing it with different ribosomal binding sites (RBSs). In total, we tested three RBSs: B0034, B0032, and strong RBS. While all three designs showed an increasing trend in fluorescence when the UV, H2O2, or nalidixic treatments were intensified, the construct with B0032 RBS showed the greatest fluorescence over OD.
FRET-based DNA damage bio-reporter system
Secondly, we are developing a DNA damage bio-reporter system using FRET. In the event of DNA damage, the generation of ssDNA spikes due to the resection of DNA from double-stranded breaks or the uncoordinated DNA unwinding and synthesis at replication forks. Extensively resected DNA ends are coated by RPA, and this ssDNA-RPA structure becomes the key structure involved in the recruitment of the ATR-ATRIP complex to sites of DNA damage (Maréchal & Zou, 2013).
While further mechanisms take place to regulate DNA damage, we are focusing on the interaction between RPA and ATR-ATRIP since it is an optimal interaction where Fluorescence resonance energy transfer (FRET) can be utilized. FRET is a distance-dependent process through which energy is transferred from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) (Sekar & Periasamy, 2003). Since its efficiency is closely related to the intermolecular separation of the target proteins, we have utilized it to detect the change in proximity of ATR-ATRIP and RPA1 when DNA damage is induced by carcinogen treatment.
By attaching a set of donor and acceptor fluorescent proteins on upstream DDR proteins (ATR-ATRIP and RPA1), the degree of energy transfer between the fluorescent proteins can be visualized. The EGFP-mCherry was selected due to its wide usage and, in terms of photophysics, its good spectral overlap. While the ECFP-EYFP pair is also common, it exhibits significant crosstalk between donor and acceptor and ECFP is sensitive to photobleaching.[16] Nonetheless, to draw a comparison, we have incorporated both pairs into our new parts ATRIP-EGFP (BBa_K4814006), RPA1-mCherry (BBa_K4814007), ATRIP-ECFP (BBa_K4814008), and RPA1-EYFP (BBa_K4814009), with EGFP and ECFP as the donor fluorophores and mCherry and EYFP as the acceptor fluorophores respectively.
hha biofilm reduction
When working with E. coli, certain contamination risks pose a threat to our and the ecosystem’s health. In addition, the extermination of E. coli requires radiation at 265 nm (the range of UVC), which is harmful to human eyes and skin. Bacterial biofilm is also involved in clinical infections and pathogenesis (Vestby et al., 2020b). In order to minimize the hazards associated with E. coli, we have utilized and tested the hha protein in our constructs, which is encoded to reduce biofilm formation and therefore the survivability of E. coli used in our experiments.
Our Aim
Rising demand for food imports drives the global genetic toxicology testing market. Demand for reagents & consumables, such as testing kits, is expected to grow fastest in the next ten years. However, mass production of these chemical assays may give rise to new environmental issues. With our innovative DNA damage reporter systems, combined with our commitment to addressing the limitations of traditional assays, we aim to offer a more effective and sustainable solution for toxicity testing.
References
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