Engineering Deinococcus Radiodurans Into Whole Cell Biosensor And The Study Of The DraSNF2 Gene

Based on a genetically modified radioresistant bacteria Deinococcus radiodurans,we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environment.The enhanced green fluorescence protein(eGFP)was fused to the promoter of a crucial DNA damage-inducible recA gene from D.radiodurans,and the consequent DNA fragment(PrecA-egfp)carried by plasmid was introduced into D.radiodurans R1 strain to obtain the biosensor strain DRG300.This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter.Based on the correlation between fluorescence intensity and protein expression level in live D.radiodurans cells,we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C.It is encouraging to find the widely detective range and high sensitivity of this reconstructed strain comparing with other whole cell biosensors in former reports.These results suggest the strain DRG300 a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.The Beta-Glo^?Assay System is a homogeneous method of quantitatingβ-galactosidase expression in mammalian cells.The system provides a bright luminescent signal that is stable over several hours in commonly used cell culture medium without prior sample processing.The homogeneous assay procedure involves the addition of a single reagent directly to cells cultured in serum-supplemented medium.Throughput rates of several thousand samples per hour may be achieved with high reproducibility under standard laboratory conditions.With this new technology,we constructed a new system for whole cell biosensor.Homologs of Snf2 family member in D.radiodurans R1,draSNF2 was sequencinged to define a single ORF including the on-line tag DR1258 and DR1259.The new defined draSNF2 gene without the intein sequence was analyzed with SSO1653,and predicted it structure and biological function.Study by treating D.radiodurans R1 strains and draSNF2 function-deficient mutant with ionizing radiation and MMC were conducted with different doses and concentration. There were no significant decreasing with the mutant's radioresistence.Based on these results, we demonstrated that draSNF2 is a member of the bacterial subfamily of Snf2 family and has different function comparing to other Snf2 members in eukaryotes.