Study On Construction And U(vi) Bioaccumulation Performance Of Recombinant D. Radiodurans-phon

Bacteria such as Citrobacter sp. harboring phoN, a gene encoding a nonspecific acid phosphatase, have been reported to precipitate heavy metals efficiently. However, the sensitivity of such bacteria to the adverse effects of radiation makes them unsuitable candidates for remediating radioactive waste. The most radiation resistant organism yet discovered is the bacterium Deinococcus radiodurans, which have the extraordinary ability to withstand radiation doses of up to 15 kG.Cloning the phoN gene into D. radiodurans by genetic engineering technology could improve host bacteria's bioaccumulation capacity in radiation environment, and achieve effective biological repair and reutilization, which has a very important scientific significance. Amplification of phoN gene, construction of recombined shuttle vector, identification of recombinant bacteria, expression of PhoN protein, and U(â…¥) bioaccumulation performance of recombinant bacteria D. radiodurans were included in this paper.PhoN gene that was amplified from Salmonella enterica serovar Typhimurium genomic DNA by PCR, was cloned into pMD18T-Vector. Recombined transfer vector T-VectorphoN was digested by restriction enzymes of Spe I and Nde I, and then the purified phoN gene was inserted into shuttle vector pRADZ3 which was digested by the same restriction enzymes. The recombined shuttle vector pRADZ3phoN was identified by PCR and restriction analysis, and transformed into E. coli DH5a and D. radiodurans competent cell. A recombinant fusion PhoN protein was expressed in normal growth condition without induction. The expression of PhoN protein in E. coli DH5a and D. radiodurans was confirmed by Western blot.The U(â…¥) bioaccumulation performance of recombinant E. coli and recombinant D. radiodurans was evaluated. The results showed that the maximum U(VI) bioaccumulation capacity of recombinant D. radiodurans increased about two times compared to original D. radiodurans, and the removal rate of U(VI) was 85.64%.It indicated that the growth of recombinant D. radiodurans was inhibited due to the inserted recombinant shuttle plasmid, compared with growth curve of recombinant D. radiodurans and original D. radiodurans. The pH value had a significant effect on U(VI) bioaccumulation performance of recombinant D. radiodurans. The removal efficiency increased with the increased of pH, and reached maximum 88.32% at pH 6. Its bioaccumulation balance time was about 60 minutes. The U(VI) bioaccumulation capacity of recombinant D. radiodurans increased with a decrease of bacteria concentration, and increased with an increase of the initial concentration of uranium.