Development of Reporter gene based Novel Bacterial Biosensors for the Detection of Arsenic and Antibiotics

Abstract

The present work was planned towards developing GFP based whole cell bacterial biosensors to aid detection studies on two serious problems facing the country i.e. rise in the prevalence of antibiotic resistant bacteria and arsenic contamination in Gangetic basin of north-eastern India. An EGFP based whole cell bacterial biosensor for arsenic detection was constructed by creating a transcriptional fusion between the ars regulatory sequences and EGFP gene in plasmid PEGFP. The resultant recombinant plasmid pJSKV51 was used to transform E. coli DH5and#945; and the new strain (E. coli DH5and#945;pJSKV51) was used as a whole cell bacterial biosensor for the detection of arsenic in drinking water samples from W. Bengal. Under optimal conditions, the Arsenic biosensor strain could detect arsenic from 5ppb to 500ppb. This range of detection satisfies the WHO standards of detection limits (50ppb) for arsenic in drinking water samples. To promote intensive studies on antibiotic resistance genes and to facilitate the selection of most suitable regulatory sequences for the construction of biosensor for antibiotic detection, an Internet based database on antibiotic resistance genes (ARGO) was developed (http;//www.argodb.org) and was made available to academia and industry as a free resource. Based on the searches made in ARGO, a bacterial biosensor strain for the detection of tetracyclines was developed by cloning the regulatory elements of tet operon from plasmid pOT182 into plasmid pEGFP. Under standard conditions, the strain could detect tetracycline in the range of 10-60ng/ml and oxytetracycline in the range of 25-125ng/ml of sample. The strain was evaluated for its efficacy by using it to detect residual tetracyclines in milk and water. The fluorescence induction in the biosensor strain (E.coliJM109pJSKV41) by all concentrations of tetracycline spiked pond water samples was equivalent to that of the EGP induction levels that was obtained by same concentrations of tetracyclines dissolved in de-ionized water. This strain was able to