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Construction of a nrdA::luxCDABE Fusion and Its Use in Escherichia coli as a DNA Damage Biosensor

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ABSTRACT

The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.

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Maximum relative luminescence values seen from strain BBTNrdA after being exposed to different concentration of (A) nalidixic acid (NDA), (B) mitomycin C (MMC), (C) 1-methyl-1-nitroso-N-methylguanidine (MNNG), and (D) 4-nitroquinoline N-oxide (4-NQO).
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f2-sensors-08-01297: Maximum relative luminescence values seen from strain BBTNrdA after being exposed to different concentration of (A) nalidixic acid (NDA), (B) mitomycin C (MMC), (C) 1-methyl-1-nitroso-N-methylguanidine (MNNG), and (D) 4-nitroquinoline N-oxide (4-NQO).

Mentions: To characterize strain BBTNrdA, we used four different chemicals that are known DNA mutagens [22], i.e., nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). Figure 2 shows the responses from this strain for each chemical. Initially, strain BBTNrdA was characterized with NDA and the maximum responses were seen at a concentration of 10 ppm (Figure 2 A), while the minimum detectable concentration (MDC) was 2.5 ppm (Table 1). The strong induction in the RBL (∼65-fold) of this strain clearly shows that this strain was responsive to NDA, which is known to inhibit the synthesis of DNA [23]. Since the nrdA genes encode for a protein specifically involved in DNA synthesis, the responses of strain BBTNrdA to NDA are not surprising.


Construction of a nrdA::luxCDABE Fusion and Its Use in Escherichia coli as a DNA Damage Biosensor
Maximum relative luminescence values seen from strain BBTNrdA after being exposed to different concentration of (A) nalidixic acid (NDA), (B) mitomycin C (MMC), (C) 1-methyl-1-nitroso-N-methylguanidine (MNNG), and (D) 4-nitroquinoline N-oxide (4-NQO).
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Related In: Results  -  Collection

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f2-sensors-08-01297: Maximum relative luminescence values seen from strain BBTNrdA after being exposed to different concentration of (A) nalidixic acid (NDA), (B) mitomycin C (MMC), (C) 1-methyl-1-nitroso-N-methylguanidine (MNNG), and (D) 4-nitroquinoline N-oxide (4-NQO).
Mentions: To characterize strain BBTNrdA, we used four different chemicals that are known DNA mutagens [22], i.e., nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). Figure 2 shows the responses from this strain for each chemical. Initially, strain BBTNrdA was characterized with NDA and the maximum responses were seen at a concentration of 10 ppm (Figure 2 A), while the minimum detectable concentration (MDC) was 2.5 ppm (Table 1). The strong induction in the RBL (∼65-fold) of this strain clearly shows that this strain was responsive to NDA, which is known to inhibit the synthesis of DNA [23]. Since the nrdA genes encode for a protein specifically involved in DNA synthesis, the responses of strain BBTNrdA to NDA are not surprising.

View Article: PubMed Central - PubMed

ABSTRACT

The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.

No MeSH data available.


Related in: MedlinePlus