DEVELOPMENT OF WHOLE CELL ARSENITE BIOSENSOR AND STUDIES ON PLASMID STABILITY IN REACTORS WITH GENETICALLY ENGINEERED CELL FOR ARSENIC BIOREMEDIATION

Abstract

The threat of arsenic pollution to public health and wild life has lead to an increased interest in developing systems that can monitor bioavailable arsenic in potable water. A highly sensitive and selective system for arsenite monitoring was developed from two genetically engineered bacteria, AW10 strain with pMV-arsR-ABS as plasmid named as modified AW10 (mAW10). Plasmid pMV-arsR-ABS provided a reduced background expression in absence of arsenite and its combination with AW10 strain provided high sensitivity at very low concentration of arsenite and to a very wide concentration range covering permissible levels. The bacterial sensing system was found to be highly selective to arsenite over a variety of metal ions and gives dose-response curve linear with coefficient of regression R2=0.9678 for a long range of arsenite concentration, 0.05 -9 uM. Our work will fetch an important contribution in prevention of diseases caused by arsenic among poor villagers who are exposed to extremely high levels of arsenic. Also our work focuses on a novel strategy, that a cell can be forced to maintain plasmid, if the plasmid is encoded with genes necessary for its survival in that environment. The cells in arsenite environment and lacking chromosomal ars operon, required for self defense against toxic environment of arsenic are forced to maintain plasmid which have arsR gene encoded on them. As arsenite is bioaccumulated by arsR proteins of the cell, this works as new defense mechanism to some extent in the cell. Cells induced by arsenite at late exponential phase were found to maintain high plasmid stability in bioreactors. The problem of plasmid stability can be over come by exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins for metal bioaccumulation under arsenic environmental pressure.