Construction Of Whole Cell Bioreporters For Genotoxicity Characterization Of Environmental Samples

Anthropogenic activities have released large amounts of potentially genotoxic chemicals into the environment. Whole cell bioreporter is a newly developed biological technology for evaluating genotoxicity both qualitatively and quantitatively, with high sensitivity, fast response and low detection limit. The whole cell bioreporter assay has provided a way for fast screen and risk assessment of environmental samples.A whole cell bioreporter E. coli recA::luxCDABE with heterogenous expressed recA from Vibrio natriegens was constructed. This bioreporter was able to evaluate genotoxicity within a wide range of external environmental conditions, with temperature from 15°C to 42°C, pH value from 4.0 to 9.0, and salinity from 0% to 3%, which has extended its application from lab to the field. The bioreporter could also assess genotoxicities in various environmental media, including surface water, groundwater, seawater, and soil matrices.Acinetobacter baylyi ADP1 is a model microorganism in soil was also used for bioreporter construction. ADP1 has a different DNA damage repair system compared with the SOS response of E. coli. Genes involved in excision repair started before recombinational repair genes. A model illuminating the process of DNA repair in ADP1 was proposed.Performance of ADP1_recA was influenced by different carbon sources, which was related to genotoxic mechanisms. The expression level of the recA promoter was influenced at the transcriptional level. Proteomics analysis showed that expression levels of 122 proteins were significantly different in defined media and LB, of which 64 were up-regulated and 58 were down-regulated. Five proteins were related to DNA-damage response, including heat shock protein 90, DNA-directed RNA polymerase, phosphate starvation-inducible protein(PhoH-like), DNA/RNA non-specific endonuclease G protein, and a two-component regulatory system response regulator.Whole cell bioreporters were applied to evaluate genotoxicities of environmental samples in various media, including groundwater from petrochemical-contaminated site and crude oil contaminated seawater. The detection limit of DH5?_V_recA_lux was as low as 0.8 ?g/L and 12.9 ng/L for VOCs and PAHs, respectively. For seawater samples, DH5?_V_recA_lux had a detection limit of ppm order of magnitude. Upon direct exposure to pre-sonicated soil samples, the bioreporters could assess genotoxicity of Cr(VI)-contaminated sites. Magnetic nanoparticle functionalized bioreporters eliminated the interference of soil matrices, and could be potentially used in genotoxicity assessment of complex media. Agar-immobilized bioreporter cells were used in a portable device for in situ genotoxicity assessment of effluents from wastewater treatment plant, which laid foundation for future application of this bioreporter assay for online environmental risk assessment.